Difference between revisions of "Team:Pasteur Paris/test/Notebook"

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quis sollicitudin lectus, a pretium erat. Donec hendrerit imperdiet
turpis, vel convallis est pharetra quis. Curabitur molestie
porttitor turpis, in fringilla arcu lacinia et.

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
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fringilla eros in ipsum molestie eleifend. Maecenas viverra ex sed
dolor blandit, at finibus orci rhoncus. Aliquam non dui est. In
mattis tincidunt nisl, at egestas felis dignissim id. Nam
condimentum accumsan tellus quis ornare. Vestibulum quis augue
malesuada, tempor lorem ut, accumsan purus. Integer nibh nulla,
malesuada ut sodales non, blandit non ligula. Donec venenatis
faucibus pulvinar. Sed tincidunt ultrices eros, nec cursus dui
molestie nec. Integer ac molestie lacus. Suspendisse lacinia mauris
in dolor ullamcorper sodales. Lorem ipsum dolor sit amet,
consectetur adipiscing elit. Pellentesque ut elit vitae neque luctus
pellentesque porta sit amet orci.
Sed eget ante ipsum. Morbi pharetra lacinia purus, vitae efficitur
eros convallis vitae. Aenean in consequat tortor, vitae dapibus
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Integer viverra orci sit amet erat fringilla, sed interdum diam
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convallis semper. Nunc nec consectetur arcu. In lectus lectus,
tempor id scelerisque vitae, egestas at purus. Curabitur vel erat
quis dui pretium lobortis nec a tellus.
Duis at efficitur sem. Suspendisse potenti. Donec pretium egestas
suscipit. Vivamus in vulputate urna. Etiam ullamcorper aliquam
blandit. Phasellus accumsan volutpat tortor non dignissim. Nunc
vestibulum tellus vel ex pulvinar convallis. Etiam ultricies
convallis malesuada. Praesent sed cursus lorem, vitae iaculis ipsum.
Vestibulum risus risus, vulputate non metus non, posuere finibus
magna. Nulla hendrerit lorem ac lacus ornare porta. Vivamus ligula
lorem, ullamcorper eu nisl eget, bibendum condimentum dui.
Aenean consectetur tincidunt ullamcorper. Vestibulum tempus, mi nec
gravida euismod, massa est consectetur ligula, vitae mattis nunc
mauris vel tortor. Vivamus ac mauris ac odio mollis volutpat vitae
ac felis. Proin eu ex et erat commodo vulputate. Morbi quis velit
eleifend libero finibus hendrerit. Mauris egestas ultrices odio in
rutrum. Pellentesque ut laoreet purus. In vitae orci ac tellus
eleifend condimentum. Phasellus at purus quis orci tempor convallis
vitae non nibh.
Cras hendrerit interdum lectus vel dictum. Donec nec lectus mattis,
aliquam tortor vitae, convallis sem. Suspendisse vitae dui tortor.
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Suspendisse
egestas, lacus a lacinia molestie, leo nibh malesuada velit, sed
iaculis nulla libero nec dui. Maecenas fringilla, massa ut pretium
hendrerit, lorem urna tristique ligula, sed pharetra eros diam vel
odio. Aenean consectetur eu massa id volutpat. Nunc vel ex laoreet,
aliquam augue ac, ullamcorper nunc. Pellentesque ut tempor dolor, ac
eleifend ante.
Pellentesque accumsan mauris in ante venenatis, nec elementum enim
ornare. In varius rutrum odio imperdiet ultricies. Pellentesque nec
feugiat tortor. Donec euismod leo orci, quis sodales felis placerat
ac. Donec a nibh dapibus, vulputate elit non, pretium nisl. Sed at
est massa. Fusce at semper tortor. In cursus lorem lectus, nec
suscipit nibh euismod eu. Morbi malesuada, lacus ut pellentesque
semper, diam tellus vulputate lectus, et varius ligula velit vitae
elit. Praesent gravida, ex sit amet ornare bibendum, metus turpis
maximus ipsum, eget interdum elit urna eu quam. Fusce tempor eu elit
id efficitur. Curabitur vitae lorem vel arcu posuere blandit at et
dolor. Nunc posuere pharetra odio vitae iaculis. In blandit dolor
quis magna cursus aliquet. Aliquam luctus hendrerit mauris ut
tristique. Vivamus pharetra scelerisque nisl vel consequat.
Integer iaculis enim et dui cursus lacinia. Fusce nec ultricies
quam, quis molestie augue. Cras mauris quam, eleifend quis sagittis
eu, venenatis non mauris. Nulla sollicitudin ut nisl at ultrices.
Vivamus fermentum placerat purus et gravida. Integer vulputate quam
nec nunc porttitor, vel gravida est aliquam. Aenean elementum eget
velit ut tincidunt. Fusce sollicitudin, magna vitae sodales
tincidunt, diam turpis fermentum urna, eget maximus est libero
lacinia nibh. Donec sagittis elementum molestie. Donec suscipit
maximus laoreet. Proin sed convallis enim. Nullam dignissim pharetra
dapibus. Aenean vel varius ipsum.
Ut eget rutrum nunc, sit amet tempor arcu. Etiam a tempus magna.
Vivamus leo nunc, consectetur non sagittis euismod, eleifend in
ante. Etiam imperdiet at dui vitae lobortis. Donec at mattis ipsum.
Nam ut ante condimentum, sodales mauris vel, tempor massa. Curabitur
feugiat libero consectetur velit viverra, nec fermentum leo
ultrices. Nulla laoreet in orci consequat condimentum. Donec et
posuere mauris, sagittis eleifend augue.
Nunc venenatis enim in auctor rutrum. Nullam molestie arcu quis
turpis mollis mattis. Phasellus elementum lectus vitae odio
tincidunt, vitae congue nisl volutpat. Fusce sodales fringilla neque
sed posuere. Morbi risus sem, imperdiet iaculis neque ac, malesuada
porta lectus. Quisque congue vel lorem vel commodo. In sit amet
gravida purus. Aliquam erat volutpat.
Praesent pellentesque pharetra sem sed condimentum. Quisque a
bibendum turpis, et malesuada ipsum. Nunc viverra quam in magna
viverra facilisis. Ut pharetra eget urna sit amet luctus. Phasellus
condimentum fermentum tortor, ut venenatis augue suscipit non.
Maecenas facilisis arcu non diam vehicula lobortis. Suspendisse ut
neque libero. Quisque sagittis iaculis faucibus. Nam sagittis,
mauris ut vestibulum tincidunt, dui ante sagittis ex, id finibus
eros augue eu velit. Maecenas vulputate ante sed hendrerit
vestibulum. Lorem ipsum dolor sit amet, consectetur adipiscing elit.
Nam enim purus, porta in leo id, accumsan gravida augue. Vestibulum
quis sollicitudin lectus, a pretium erat. Donec hendrerit imperdiet
turpis, vel convallis est pharetra quis. Curabitur molestie
porttitor turpis, in fringilla arcu lacinia et.

July Week 4 : 07/22 to 07/28

07.22.19
Resuspension of oligonucleotides :
DNA library and primers were produced by Eurofins Genomics. Once received, they were resuspended according to the manufacturer’s instructions. Stock solutions of 100 µM were made by diluting the lyophilized oligonucleotides with a 10 mM Tris-EDTA pH 8.0 solution. Concentrations were checked using a Nanodrop 2000 spectrophotometer.

Samples
Concentration (ng/µl)
260/280 ratio
260/230 ratio

DNA library
2217.65
1.89
1.56

Reverse primer
8810.9
1.95
1.58

PCR 1 - Amplification of the DNA library
In order to start the SELEX, we need to amplify our DNA library in order to get enough material to start with. We tried several PCR protocols to optimize the amplification. This test was performed in order to know if the magnesium concentration could impact the quality of the amplification, and different annealing temperatures from 44 to 54°C were tested. A mix of Taq DNA polymerase and phusion DNA polymerase was used.

Volume needed (μL)

Components
Final concentration
Mix 1
Mix 2
Mix 3

Phusion HF buffer 5X
1X
50
50
50

25 mM dNTPs
0.20 mM
2
2
2

Forward primer
0.5 μM
9.75
9.75
9.75

Reverse primer
0.5 μM
10.1
10.1
10.1

25 mM Magnesium
2.5 mM
25
50
75

DNA library
10 ng
22.5
22.5
22.5

Taq DNA polymerase
0.1 U/μL
0.02
0.02
0.02

Phusion DNA polymerase
1 U/μL
0.5
0.5
0.5

Nuclease-free water
/
130.13
105.13
80.13

Total volume
/
250
250
250

Rq: Enzymes were added at the end as hot-start PCR protocols can optimize the yield of the desired product while limiting the likelihood of nonspecific amplification

PCR plate plan

A (54°C)
1.1
2.1
3.1

B
/
/
/

C (51.9°C)
1.2
2.2
3.2

D
/
/
/

E (47.6°C)
1.3
2.3
3.3

F
/
/
/

G (44.7°C)
1.4
2.4
3.4

G
/
/
/

PCR program used:

- 95°C 5 min

- 95°C 30 s

- 44-54°C 1 min 30 cycles

- 68°C 30 s

- 68°C 5 min

- 15°C HOLD ALL NIGHT

PCR was run overnight. Samples were checked using a Nanodrop 2000 spectrophotometer.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ratio

1.1
465.9
1.39
0.66

1.2
501.7
1.42
0.69

1.3
480.4
1.43
0.68

1.4
469.9
1.43
0.65

2.1
498.3
1.40
0.68

2.2
498.7
1.43
0.70

2.3
532.9
1.42
0.71

2.4
495.2
1.42
0.69

3.1
497.8
1.43
0.70

3.2
512.2
1.43
0.70

3.3
505.5
1.42
0.70

3.4
527.2
1.41
0.70

Interpretations :

No special condition seemed better than others.

07.23.19

Gel electrophoresis 1 - Amplified library

To verify that the samples were all the same with the right size, we performed a gel electrophoresis using a 3% agarose gel. Each band was cut and then kept at -4°C in order to be purified later.

1.
Sample 1.1 (54°C, 25 μL, Mg2+)

2.
Sample 1.2 (51.9°C, 25 μL, Mg2+)

3.
Sample 1.3 (47.6°C, 25 μL, Mg2+)

4.
Sample 1.4 (44.7°C, 25 μL, Mg2+)

5.
Sample 2.1 (54°C, 50 μL, Mg2+)

6.
Sample 2.2 (51.9°C, 50 μL, Mg2+)

7.
Sample 2.3 (47.6°C, 50 μL, Mg2+)

8.
Sample 2.4 (44.7°C, 50 μL, Mg2+)

9.
Sample 3.1 (54°C, 75 μL, Mg2+)

10.
Sample 3.2 (51.9°C, 75 μL, Mg2+)

11.
Sample 3.3 (47.6°C, 75 μL, Mg2+)

12.
Sample 3.4 (44.7°C, 75 μL, Mg2+)

Interpretations

All samples had the same size of 120 bp approximately whereas the library size was 80 bp normally.

Conclusion

PCR amplification resulted in a shift in size of the oligonucleotides because of the double-stranded form.

Cell culture

TO COMPLETE

07.24.19

Growth curves

Bacteria were cultured and their growth was analyzed by establishing a growth curve associated with the number of colonies. Several strains were used:

E. Faecium (ref CIP 103014T)
P. Aeruginosa (ref 104116)
A. Baumanii (ref CIP 79.34T)
S. Aureus (ref CIP 103429)
S. Pneumoniae (ref CIP 104340)
E. Coli (ref 53.126)
E. Enterica (ref CIP 60.62T)

All strains were provided by the CRBIP of the Pasteur Institute.

TO COMPLETE

07.25.19

Purification of PCR products - Invitrogen Pure Link Quick Gel extraction kit - PCR 1

After PCR amplification, PCR products need to be cleaned up. Gel slices were extracted and purified using Invitrogen Pure Link Quick Gel extraction kit following the manufacturer’s instructions.

END OF THE WEEK

July Week 5 : 07/29
to 08/04

07.29.19
Gel electrophoresis 2 - Purified PCR products from gel extraction kit

To verify that the samples were purified after gel extraction, we performed a gel electrophoresis using a 3% agarose gel. We used two ladders, one at 25 bp and the other at 50 bp from Promega.

Interpretation

There was no band after purification by gel extraction. So, during the purification step we lost the DNA.

Digestion - Amplified library

This step is made to pass from a double-stranded DNA to a single-stranded one.
We performed the digestion using Lambda Exonuclease from New England Biolabs directly on the amplified library (PCR 1) without purifying it beforehand.

1. Set-up the reaction as follows:

Components
Volume for 50 μL reaction

DNA from PCR 1
5 μg

Lambda Exonuclease Reaction Buffer (10X)
5 μL (1X)

Lambda Exonuclease
1 μL (5 units)

Nuclease-free water
up to 50μL

2. Incubate at 37°C for 30 minutes.

3. Stop reaction by adding EDTA to 10 mM.

4. Heat at 75°C for 10 minutes.

07.31.19

PCR 2 - Testing different conditions

To try to enhance our PCR protocol, we performed a new PCR using different conditions. These conditions enabled us to determine if the Taq DNA Polymerase has to be activated prior addition, and if both polymerases have to be added at 95°C or not. The PCR was run overnight and hold at 4°C.

Volume needed (in μL)

Components
Mix 1
Mix 2

Phusion HF buffer 5X
500
500

25 mM dNTPs
20
250

Forward primer
97.5
50

Reverse primer
101
50

25 mM Magnesium
250
250

DNA library (1/1000 dilution)
225
250

Taq DNA polymerase diluted 1/10
5
5

Phusion DNA polymerase
25
25

Nuclease-free water
1276.5
1135

Total volume
2500
2500

PCR plan

1
2
3
4
5
6
7
8
9
10
11
12

A
1A
2A
3A
4A
5A
6A
7A
8A
9A
10A
11A
12A

B
1B
2B
3B
4B
5B
6B
7B
8B
9B
10B
11B
12B

C
1C
2C
3C
4C
5C
6C
7C
8C
9C
10C
11C
12C

D
1D
2D
3D
4D
5D
6D
7D
8D
9D
10D
11D
12D

E
1E
2E
3E
4E
5E
6E
7E
8E
9E
10E
11E
12E

F
1F
2F
3F
4F
5F
6F
7F
8F
9F
10F
11F
12F

G
1G
2G
3G
4G
5G
6G
7G
8G
9G
10G
11G
12G

H
1H
2H
3H
4H
5H
6H
7H
8H
9H
10H
11H
12H

Mix 1
Mix 2

Legend :

Condition 1: Taq was activated and added with Phusion in ice
Condition 2: Taq was not activated and added with Phusion in ice
Condition 3: Phusion was added in ice
Condition 4: Taq was activated and added with Phusion at 95°C
Condition 5: Taq was not activated and added with Phusion at 95°C
Condition 6: Phusion was added at 95°C
Condition 7: Taq was activated and added with Phusion in ice
Condition 8: Taq was not activated and added with Phusion in ice
Condition 9: Phusion was added in ice
Condition 10: Taq was activated and added with Phusion at 95°C
Condition 11: Taq was not activated and added with Phusion at 95°C
Condition 12: Phusion was added at 95°C

Gel electrophoresis 4 - PCR 2

A gel containing 3% of agarose was run to determine which condition led to the best results.

Interpretation

Condition 1 provided the best results because the band is the brightest. Moreover, there was almost no difference between the samples containing activated Taq polymerase and when the polymerases were added on ice or in 95°C. Because we obtained the same results when we amplified using both Taq and phusion polymerases and only Phusion polymerase, we decided to only use Phusion polymerase for our PCR.

08.01.19

DNA precipitation - Product of phenol/chloroform from 29.07.19

1. Add 1/10 volume of Sodium Acetate (3 M, pH 5.2) and 2,5x volume of 100% ethanol

Sample
Pool 1 Elution 1
Pool 1 Elution 2
Pool 2 Elution 1
Pool 2 Elution 2

Upper aqueous phase recovered
2000 µL
430 µL
1150 µL
415 µL

Sodium acetate 3M to add
200 µL
43 µL
115 µL
41.5 µL

Volume after addition of sodium acetate
2200 µL
473 µL
1265 µL
456.5 µL

Ethanol 100% to add
5500 µL
1182.5 µL
3162.5 µL
1141.25 µL

Total volume
7700 µL
1655.5 µL
4427.5 µL
1597.75 µL

2. Place the tube at -20°C overnight to precipitate the DNA from the sample. The experiment was continued on 08.02.19

08.2.19

DNA precipitation

1. Centrifuge the sample at 4°C for 30 minutes at 16,000 × g to pellet the cDNA.
2. Carefully remove the supernatant without disturbing the cDNA pellet.
3. Add 150 μL of 70% ethanol.
4. Centrifuge the sample at 4°C for 15 minutes at 16,000 × g. Carefully remove the supernatant.
5. Dry the cDNA pellet at room temperature
6. Resuspend the cDNA pellet in 300 μL of TEN buffer by pipetting up and down

Purification of PCR product - illustra MicroSpin G-25 Columns kit from GE Healthcare - PCR 2

Because the purification by gel extraction was not conclusive, we tested a second method of purification using the illustra MicroSpin G-25 Columns kit from GE Healthcare. The experiment was performed according to the manufacturer’s recommendation.

Gel electrophoresis 5 - Purified products from gel extraction vs. columns

Because the purification by gel extraction was not conclusive, we tested a second method of purification using the illustra MicroSpin G-25 Columns kit from GE Healthcare. The experiment was performed according to the manufacturer’s recommendation.

Interpretation

We only observed the bands from the samples purified with the columns. Moreover, they seemed well purified because there was almost no smear.

PCR 3 - Testing different conditions (with/without DMSO + new program)

To try to enhance our PCR protocol, we performed a new PCR using two conditions, one with DMSO and the other without . The PCR was run overnight and hold at 4°C.

Volume needed (in μL)

Components
Mix 1 (With DMSO)
Mix 2 (Without DMSO)

Phusion HF buffer 5X
50
50

25 mM dNTPs
5
5

Forward primer
10
10

Reverse primer
10
10

DMSO
7.5
0

Template DNA
22.5
22.5

Phusion DNA polymerase
2.5
2.5

Nuclease-free water
142.5
150

Total volume
250
250

August Week 1 : 08/05 to 08/11

08.05.19

Purification of PCR products - illustra MicroSpin G-25 Columns kit from GE Healthcare - PCR 3

Once again, we tried to purify our PCR products using different methods in order to compare them. This experiment was performed according to the manufacturer’s protocol.

Purification of PCR products - PCR clean-up kit from Quiagen - PCR 3

This experiment was performed according to the manufacturer’s protocol.

Gel electrophoresis 6 - Purified products from gel extraction (30.07.19) vs. GE Healthcare vs. Quiagen

To analyze the different purification methods of our PCR products from 02.08 and the effect of DMSO, we ran the samples on an agarose gel. Moreover, we compared the percentage of agarose in gel between 3 and 4% to see if the band resolution was better.

1. PCR product from 02.08 with DMSO
2. PCR product from 02.08 without DMSO
3. PCR product with DMSO purified with Qiagen kit
4. PCR product whithout DMSO purified with Qiagen kit
5. PCR product with DMSO purified with GE Healthcare column
6. PCR product without DMSO purified with GE Healthcare column
7. PCR product with DMSO purified by gel extraction
8. PCR product without DMSO purified by gel exctraction

Wells
Purification method
Concentration (ng/μL)
260/280 ratio
260/230 ratio

4
PCR product with DMSO
1157.6
1.64
0.85

5
PCR product without DMSO
1143
1.65
1.18

6
PCR clean up kit from QIAGEN with DMSO
7.9
2.26
-0.32

7
PCR clean up kit from QIAGEN without DMSO
15
2.07
-0.62

8
GE Healthcare columns with DMSO
34.5
0.96
0.34

9
GE Healthcare columns without DMSO
45.9
1.16
0.08

10
Gel extraction kit with DMSO
6.1
-6.66
0.01

11
Gel extraction kit without DMSO
-0.5
0.53
0

Interpretations

The gel containing 4% of agarose gave a better resolution of the bands. Moreover, we did not observe any difference between the amplified products with and without DMSO. Concerning the purification methods, there was still no bands after the purification by gel extraction. Concerning the purification by PCR clean-up, we did not see anything on the gel at 4% agarose and there was only one band in the well 9 on the gel with 3% of agarose. On the contrary, we observed a band on both gels for the two products purified by the columns.

We analyzed then the same products as above but after digestion. The digestion was performed according to the manufacturer’s protocol. This time we only did a 4% of agarose gel because we noticed with the previous experiment that we had a better resolution with this concentration.

Interpretation

After digestion, there was no more bands even for the sample purified with the columns. Thus, either the lambda exonuclease had digested the two strands of DNA, or the concentration of DNA was too low to be seen on the gel. The last hypothesis was that the SYBR Green may not reveal single-stranded DNA.

Conclusion

Several parameters may change during the digestion step in order to determine if the enzyme digested the two strands of DNA.

Phenol/chloroform extraction

We performed a purification step on all samples to remove the digestion enzyme according to the protocol previously described. Samples were then analyzed using a Nanodrop 2000 spectrophotometer.

Wells
Purification method
Concentration (ng/μL)
260/280 ratio
260/230 ratio

5
PCR product with DMSO
1157.6
1.64
0.85

6
PCR product without DMSO
1143
1.65
1.18

8
PCR product clean up kit from QIAGEN with DMSO
7.9
2.26
-0.32

9
PCR product clean up kit from QIAGEN without DMSO
15
2.07
-0.62

10
GE Healthcare columns with DMSO
34.5
0.96
0.34

11
GE Healthcare columns without DMSO
45.9
1.16
0.08

13
Gel extraction kit with DMSO
6.1
-6.66
0.01

14
Gel extraction kit without DMSO
-0.5
0.53
0

07.08.19

PCR 4 - 15 cycles vs 30 cycles

We tried to amplify our library with only 15 cycles instead of 30 to compare if it had an impact in the shift of size of our PCR product (120 bp instead of 80 bp normally). The PCR was made with the same conditions as the PCR 4 without DMSO.

Gel electrophoresis 7 - 15 cycles vs 30 cycles

The amplified products were run on a 4% agarose gel and compared with amplified products from PCR 3 and 4.

Interpretations

We observed a slight decrease of the size for products amplified with only 15 cycles compared to those amplified with 30 cycles. However, this decrease in size was not significant enough. And the bands from the amplified samples with 30 cycles were brighter than those amplified with 15 cycles.

Purification of PCR products - Dialysis tubing

A new purification method of our PCR products was tested which is dialysis tubing. The DNA will migrate out of the gel and into the dialysis bag. Samples were checked using a Nanodrop 2000 spectrophotometer.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ratio

Dialysis sample 1 pool
1.2
2.71
-9.20

Dialysis sample 2 pool
2.8
2.22
0.6

Interpretation

Even if the ratio 260/280 was good the concentration of DNA was too low. Thus, this technique cannot be used due to a yield too low.

Kit montage Gel Extraction

We used the Kit montage Gel extraction from Millipore to try to extract our DNA from the gel and purify it. This kit was used according to the manufacturer’s protocol. Samples were checked using a Nanodrop 2000 spectrophotometer.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ratio

Sample 1
8.3
1.95
0.15

Sample 2
6.4
2.13
0.16

Sample 3
5
2.24
0.12

Sample 4
6.9
1.37
0.14

Interpretation

The concentration of DNA was too low. Hence, another method of DNA purification has to be found.

August Week 2 : 07/12 to 07/18
07.12.19
Digestion - Testing different conditions on
amplified library
A new digestion was carried out in order to optimize the
yield. Two parameters were studied, the incubation time and the
quantity of enzyme used. Here is the list of all conditions tested:
1A : 5 min incubation, 5 U
1B : 5 min incubation, 2.5 U
1C : 5 min incubation, 1 U
2A : 10 min incubation, 5 U
2B : 10 min incubation, 2.5 U
2C : 10 min incubation, 1 U
3A : 15 min incubation, 5 U
3B : 15 min incubation, 2.5 U
3C : 15 min incubation, 1 U
4A : 20 min incubation, 5 U
4B : 20 min incubation, 2.5 U
4C : 20 min incubation, 1 U

Gel electrophoresis 8 - Digestion conditions
comparison

Samples were then checked using a Nanodrop 2000
spectrophotometer.

Sample
Concentration (ng/µl)
260/280 ratio
260/230 ratio

1A after digestion
121.4
1.52
0.29

1B after digestion
117.2
1.52
0.29

1C after digestion
118
1.55
0.29

2A after digestion
69.3
1.64
0.3

2B after digestion
116.5
1.49
0.3

2C after digestion
117.3
1.51
0.28

3A after digestion
119
1.55
0.28

3B after digestion
110.6
1.54
0.28

3C after digestion
117.4
1.57
0.24

4A after digestion
120.5
1.52
0.26

4B after digestion
118.3
1.56
0.24

4C after digestion
130.1
1.52
0.26

Interpretations

There was no bands on the gel. However, the nanodrop detected DNA with a pretty good 260/280 ratio. But there was no difference between the different conditions. So, either the single-strand DNA i s not revealed with SYBR Green or the digestion enzyme does not work properly.

08.14.19

Denaturation - of PCR 5 products

In order to optimize the digestion step, we tried to denature our PCR product before being digested. Samples were heated at 95°C for 5 min and directly put in ice for 15 min.

Digestion - Testing different conditions on PCR 5 products

Different conditions of digestion were tested in order to optimize this step (buffers, DNA quantity to digest, enzyme quantity, with/without denaturation before).
Conditions tested:

Cutsmart, 15 µg DNA, 1 U (37°C, 15 min)
Cutsmart, 10 µg DNA, 1 U (37°C, 15 min)
Cutsmart, 15 µg DNA, 0,5 U (37°C, 15 min)
Cutsmart, 10 µg DNA, 0,5 U (37°C, 15 min)
Lambda buffer, 15 µg DNA, 1 U (37°C, 15 min)
Lambda buffer, 10 µg DNA, 1 U (37°C, 15 min)
Lambda buffer, 15 µg DNA, 0,5 U (37°C, 15 min)
Lambda buffer, 10 µg DNA, 0,5 U (37°C, 15 min)
Lambda buffer, 15 µg DNA, 1 U (37°C, 15 min) + denaturation before
Lambda buffer, 10 µg DNA, 1 U (37°C, 15 min) + denaturation before
Lambda buffer, 15 µg DNA, 0,5 U (37°C, 15 min) + denaturation before
Lambda buffer, 10 µg DNA, 0,5 U (37°C, 15 min) + denaturation before
50 bp ladder
PCR product before digestion
PCR product before digestion followed by heat shock

Gel electrophoresis 9 - Digestion conditions comparison

A 4% agarose gel was made to compare all the different conditions of digestion.

Interpretations

We are looking for a band with a shift in size compared to the PCR product before digestion. Once again, bands were similar to the PCR product, it means that conditions were too restrictive and that the digestion did not worked.

08.15.19

PCR 6 - Generation of new materials

To continue our experiments, a new part of the library was amplified in order to work with. Samples were also analyzed using a Nanodrop 2000 spectrophotometer.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ratio

Sample 1
1231.1
1.65
1.15

Sample 2
1217.1
1.5
1.15

Sample 3
1219.1
1.66
1.16

Sample 4
1215.3
1.64
1.15

Sample 5
1216.2
1.65
1.15

Sample 6
1213.2
1.66
1.14

Sample 7
1201.4
1.66
1.14

Sample 8
1211.5
1.65
1.15

Sample 9
1221.2
1.65
1.14

Sample 10
1185.2
1.65
1.14

August Week 3 : 08/19 to
08/21

08.19.19
PCR 7 - Generation of new materials (10 ng, 5 ng, 1
ng)
A new PCR was performed to produce starting material. For
this, different amounts of DNA to be amplified have been tested (10
ng, 5 ng, 1 ng) in order to reduce nonspecific amplifications.

Volume needed (in μL)

Components
Mix 1(10ng)
Mix 2(5ng)
Mix 3(1ng)

Phusion Buffer 10X
100
100
100

dNTPs 25 mM
10
10
10

Forward primer (1/10)
20
20
20

Reverse primer (1/10)
20
20
20

Template DNA (1/1000)
45
22.5
5

Phusion DNA polymerase
5
5
5

Nuclease-free water
300
322.5
340

Total volume
500
500
500

Samples were also analyzed using a Nanodrop 2000
spectrophotometer.

Sample
Concentration (ng/μl)
260/280 ratio
260/230 ratio

Sample pooled (10ng)
990.6
1.62
1.05

Sample pooled (5ng)
1028.5)
1.61
1.05

Sample pooled (1ng)
1028.7)
1.61
1.04

08.20.10
Digestion - 30 min, 5 U, from 5 to 1 µg with control
without enzyme from PCR 7
A new digestion was performed with different quantity of
DNA to digest for each sample. An undigested sample from the PCR 7 was
used as a negative control.
Gel electrophoresis 10 - Digestion conditions comparison

A gel with 4% of agarose was made to compare each sample.
The staining was performed with GelRed because it is more specific of
single-strand DNA than ethidium bromide and SYBR Green.

08.21.19
PCR 8 - Generation of new materials
A new PCR was performed to produce starting material.

Samples were analyzed using a Nanodrop 2000 spectrophotometer.

Sample
Concentration(ng/µl
260/280 ratio
260/230 ratio

PCR 8 pooled
1093.9
1.63
1.11

August Week 4 : 08/26 to
09/1

08.26.19

Purification of PCR product - illustra
MicroSpin G-25 Columns kit from GE Healthcare - PCR 9
The PCR 9 products were purified according to the
manufacturer’s protocol. Then, samples were analyzed using a Nanodrop
2000 spectrophotometer.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ratio

PCR 9 purified by column
868.7
1.58
1.03

Digestion - from PCR 9
After the purification, the sample was digested
according to the manufacturer’s instructions with 4 µg of DNA.

Phenol/chloroform extraction - Digested
products from PCR 9
The sample was purified by phenol/chloroform according
to the protocol previously described. Then, samples were analyzed
using a Nanodrop 2000 spectrophotometer.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ration

PCR 9 purified by phenol/chloroform
17
1.48
1.34

Interpretation
We lose a huge quantity of DNA. Thus, the concentration
is too low to begin the first round of SELEX.

08.27.19

Digestion - from PCR 9
We purify the digested samples using either 2.5 X volume
of ethanol, as previously, or 1 X volume of isopropanol. The samples
were then analyzed using a Nanodrop 2000 spectrophotometer.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ration

PCR 9 purified with ethanol 1
9.2
1.6
0.91

PCR 9 purified with ethanol 2
7.3
1.5
0.91

PCR 9 purified with isopropanol 1
44.9
1.41
1.10

PCR 9 purified with isopropanol 1
48.1
1.42
1.39

Interpretations
The concentration of DNA are much higher when we used
isopropanol compared to ethanol.

Conclusion
Isopropanol is used for the next purifications by
phenol/chloroform. These concentrations are enough to perform the
first round of SELEX on one bacteria. However, we want to select
aptamers for two different bacteria so we must perform this step
again.

08.28.19
Bacteria culture - E. faecium and S. aureus

The strains E. faecium and S. aureus were grown on Petri
dish on Blood agar and LB media respectively. The plates were
incubated at 37°C during 24 hours.

Digestion - from PCR9
The PCR 9 products were digested using 4 µg of DNA
according to the manufacturer’s protocol.
Phenol/chloroform extraction with Isopropanol
- Digested products from PCR 9
After digestion, the samples were purified using 1 X
volume of isopropanol. The samples are then analysed using a Nanodrop
2000 spectrophotometer.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ration

PCR 9 purified by phenol/chloroform
1
42.1
1.46
1.40

PCR 9 purified by phenol/chloroform 2
3.1
1.65
0.71

PCR 9 purified by phenol/chloroform 3
3.1
1.55
1.64

PCR 9 purified by phenol/chloroform 4
8.6
1.43
1.19

Interpretations
The concentration of DNA is lower than expected. We may
have pipeted some DNA as the pellet is invisible.

Conclusion
Because of these low concentrations, the first round of
SELEX cannot be performed.

08.29.19

Bacteria culture - E. faecium and S. aureus

Few colonies of E. faecium and S. aureus were taken from
the Petri dish culture of the 28.08 and grow in their respective
liquid media at 37°C under agitation. After 3 hours of growth, they
are centrifuged and the pellet of bacteria is resuspended in PBS 1X.
Dilutions were performed until the OD reached between 0.5 and 0.6.

Digestion - from PCR9
In order to have more DNA before starting the SELEX, we
performed a digestion with 4 µg of DNA from the PCR 9 samples
according to the manufacturer’s protocol.

Phenol/chloroform extraction with Isopropanol
- Digested products from PCR 9
The samples were purified after digestion using 1 X
volume of isopropanol. Then, they were analysed with a Nanodrop 2000
spectrophotometer.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ration

PCR 9 purified sample 1
41.4
1.41
1.46

PCR 9 purified sample 2
4.2
1.25
0.57

PCR 9 purified sample 3
9.4
1.33
0.99

PCR 9 purified sample 4
23.7
1.39
1.08

PCR 9 purified sample 5
11.1
1.37
0.41

PCR 9 purified sample 6
27.8
1.40
0.90

Conclusion

After the purification, we have enough DNA concentration to start the SELEX on the two bacteria.

SELEX 1 Round 1

The first round of SELEX was performed with 2 hours of incubation. The samples are then analyzed using a Nanodrop 2000 spectrophotometer.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ration

S. aureus
60.1
1.61
0.96

E. faecium
43.6
1.69
1.09

PCR 10 - Amplification of EFr1 & SAr1

This PCR was performed to amplified the DNA after the first round of SELEX. We tried two different concentrations for both samples. The PCR followed the same program as described previously. It was run overnight and hold at 4°C.

S aureus

Components
SA 1 (1/10)
SA 2 (1/90)

Phusion buffer
10
10

dNTPs
1
1

Forward primer
2
2

Reverse primer
2
2

Template DNA (1/10)
1
0

Template DNA (1/90)
0
1

Phusion DNA polymerase
0.5
0.5

Nulease-free water
33.5
33.5

Total volume
50
50

E. faecium

Components
SA 1 (1/10)
SA 2 (1/80)

Phusion buffer
10
10

dNTPs
1
1

Forward primer
2
2

Reverse primer
2
2

Template DNA (1/10)
1
0

Template DNA (1/80)
0
1

Phusion DNA polymerase
0.5
0.5

Nulease-free water
33.5
33.5

Total volume
50
50

The samples are analyzed using a Nanodrop 2000 spectrophotometer.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ration

PCR 10 - Round 1 - SA 1
1253.9
1.62
1.05

PCR 10 - Round 1 - SA 2
1200.4
1.64
1.09

PCR 10 - Round 1 - EF 1
1118.9
1.56
0.89

PCR 10 - Round 1 - EF 2
1207.9
1.66
1.13

08.30.19

Gel electrophoresis 12 - PCR 10

On a 4% agarose gel, samples before and after the PCR 12 were run.

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
purus augue, dictum non molestie eget, posuere a mauris. Donec
fringilla eros in ipsum molestie eleifend. Maecenas viverra ex sed
dolor blandit, at finibus orci rhoncus. Aliquam non dui est. In
mattis tincidunt nisl, at egestas felis dignissim id. Nam
condimentum accumsan tellus quis ornare. Vestibulum quis augue
malesuada, tempor lorem ut, accumsan purus. Integer nibh nulla,
malesuada ut sodales non, blandit non ligula. Donec venenatis
faucibus pulvinar. Sed tincidunt ultrices eros, nec cursus dui
molestie nec. Integer ac molestie lacus. Suspendisse lacinia mauris
in dolor ullamcorper sodales. Lorem ipsum dolor sit amet,
consectetur adipiscing elit. Pellentesque ut elit vitae neque luctus
pellentesque porta sit amet orci.
Sed eget ante ipsum. Morbi pharetra lacinia purus, vitae efficitur
eros convallis vitae. Aenean in consequat tortor, vitae dapibus
nisi. Nunc non imperdiet tortor. Curabitur aliquet facilisis tempus.
Integer viverra orci sit amet erat fringilla, sed interdum diam
euismod. Ut ac vulputate orci. Nunc iaculis tellus quis massa
convallis semper. Nunc nec consectetur arcu. In lectus lectus,
tempor id scelerisque vitae, egestas at purus. Curabitur vel erat
quis dui pretium lobortis nec a tellus.
Duis at efficitur sem. Suspendisse potenti. Donec pretium egestas
suscipit. Vivamus in vulputate urna. Etiam ullamcorper aliquam
blandit. Phasellus accumsan volutpat tortor non dignissim. Nunc
vestibulum tellus vel ex pulvinar convallis. Etiam ultricies
convallis malesuada. Praesent sed cursus lorem, vitae iaculis ipsum.
Vestibulum risus risus, vulputate non metus non, posuere finibus
magna. Nulla hendrerit lorem ac lacus ornare porta. Vivamus ligula
lorem, ullamcorper eu nisl eget, bibendum condimentum dui.
Aenean consectetur tincidunt ullamcorper. Vestibulum tempus, mi nec
gravida euismod, massa est consectetur ligula, vitae mattis nunc
mauris vel tortor. Vivamus ac mauris ac odio mollis volutpat vitae
ac felis. Proin eu ex et erat commodo vulputate. Morbi quis velit
eleifend libero finibus hendrerit. Mauris egestas ultrices odio in
rutrum. Pellentesque ut laoreet purus. In vitae orci ac tellus
eleifend condimentum. Phasellus at purus quis orci tempor convallis
vitae non nibh.
Cras hendrerit interdum lectus vel dictum. Donec nec lectus mattis,
aliquam tortor vitae, convallis sem. Suspendisse vitae dui tortor.
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Suspendisse
egestas, lacus a lacinia molestie, leo nibh malesuada velit, sed
iaculis nulla libero nec dui. Maecenas fringilla, massa ut pretium
hendrerit, lorem urna tristique ligula, sed pharetra eros diam vel
odio. Aenean consectetur eu massa id volutpat. Nunc vel ex laoreet,
aliquam augue ac, ullamcorper nunc. Pellentesque ut tempor dolor, ac
eleifend ante.
Pellentesque accumsan mauris in ante venenatis, nec elementum enim
ornare. In varius rutrum odio imperdiet ultricies. Pellentesque nec
feugiat tortor. Donec euismod leo orci, quis sodales felis placerat
ac. Donec a nibh dapibus, vulputate elit non, pretium nisl. Sed at
est massa. Fusce at semper tortor. In cursus lorem lectus, nec
suscipit nibh euismod eu. Morbi malesuada, lacus ut pellentesque
semper, diam tellus vulputate lectus, et varius ligula velit vitae
elit. Praesent gravida, ex sit amet ornare bibendum, metus turpis
maximus ipsum, eget interdum elit urna eu quam. Fusce tempor eu elit
id efficitur. Curabitur vitae lorem vel arcu posuere blandit at et
dolor. Nunc posuere pharetra odio vitae iaculis. In blandit dolor
quis magna cursus aliquet. Aliquam luctus hendrerit mauris ut
tristique. Vivamus pharetra scelerisque nisl vel consequat.
Integer iaculis enim et dui cursus lacinia. Fusce nec ultricies
quam, quis molestie augue. Cras mauris quam, eleifend quis sagittis
eu, venenatis non mauris. Nulla sollicitudin ut nisl at ultrices.
Vivamus fermentum placerat purus et gravida. Integer vulputate quam
nec nunc porttitor, vel gravida est aliquam. Aenean elementum eget
velit ut tincidunt. Fusce sollicitudin, magna vitae sodales
tincidunt, diam turpis fermentum urna, eget maximus est libero
lacinia nibh. Donec sagittis elementum molestie. Donec suscipit
maximus laoreet. Proin sed convallis enim. Nullam dignissim pharetra
dapibus. Aenean vel varius ipsum.
Ut eget rutrum nunc, sit amet tempor arcu. Etiam a tempus magna.
Vivamus leo nunc, consectetur non sagittis euismod, eleifend in
ante. Etiam imperdiet at dui vitae lobortis. Donec at mattis ipsum.
Nam ut ante condimentum, sodales mauris vel, tempor massa. Curabitur
feugiat libero consectetur velit viverra, nec fermentum leo
ultrices. Nulla laoreet in orci consequat condimentum. Donec et
posuere mauris, sagittis eleifend augue.
Nunc venenatis enim in auctor rutrum. Nullam molestie arcu quis
turpis mollis mattis. Phasellus elementum lectus vitae odio
tincidunt, vitae congue nisl volutpat. Fusce sodales fringilla neque
sed posuere. Morbi risus sem, imperdiet iaculis neque ac, malesuada
porta lectus. Quisque congue vel lorem vel commodo. In sit amet
gravida purus. Aliquam erat volutpat.
Praesent pellentesque pharetra sem sed condimentum. Quisque a
bibendum turpis, et malesuada ipsum. Nunc viverra quam in magna
viverra facilisis. Ut pharetra eget urna sit amet luctus. Phasellus
condimentum fermentum tortor, ut venenatis augue suscipit non.
Maecenas facilisis arcu non diam vehicula lobortis. Suspendisse ut
neque libero. Quisque sagittis iaculis faucibus. Nam sagittis,
mauris ut vestibulum tincidunt, dui ante sagittis ex, id finibus
eros augue eu velit. Maecenas vulputate ante sed hendrerit
vestibulum. Lorem ipsum dolor sit amet, consectetur adipiscing elit.
Nam enim purus, porta in leo id, accumsan gravida augue. Vestibulum
quis sollicitudin lectus, a pretium erat. Donec hendrerit imperdiet
turpis, vel convallis est pharetra quis. Curabitur molestie
porttitor turpis, in fringilla arcu lacinia et.

September Week 1 : 09/05 to 09/15

Digestion - EFr1 & SAr1

The digestion was performed with 4 µg of DNA according to the manufacturer’s protocol.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ratio

EFr1 digested
27.4
1.87
0.26

SAr1 digested
30.2
1.89
0.28

Purification of digested products EFr1 & SAr1 - kit Monarch from NEB

A new kit of purification was tried to purify the PCR Products. The experiment was performed according to the manufacturer’s recommendations.

The samples are checked using a Nanodrop 2000 spectrophotometer.

Sample
Concentration (ng/µl)
260/280 ratio
260/230 ratio

EFr1 purified
7.8
1.27
0.27

EFr1 wash 1
-1.7
0.58
-0.03

EFr1 wash 2
2.5
5.51
0.27

EFr1 purified
7.8
1.27
0.27

SAr1 purified
7.2
1.52
0.58

SAr1 wash 1
22.5
1.29
0.53

SAr1 wash 2
2.4
8.31
0.30

Interpretations

The concentrations are really low. Moreover, the quantity of DNA within the sample is not high because the ratio 260/280 is not close to 2. Finally, the ratio 260/230 did not change increase before and after the purification.

Conclusion

This kit cannot be used to purify our DNA after digestion.

Purification of digested products EFr1 & SAr1 - illustra MicroSpin G-25 Columns kit from GE Healthcare

The purification was performed according to the manufacturer’s protocol.
The samples are checked using a Nanodrop 2000 spectrophotometer.

Sample
Concentration (ng/μL)
260/280 ratio
260/230 ratio

EFr1 purified
53.2
1.62
0.24

SAr1 purified
56.2
1.54
0.27

Interpretation

Even if the concentrations are quite elevated, we noticed that the ratio 260/230 did not change before and after the purification using these columns. Hence, the enzymes may not be purified by this technique.

Conclusion

We tried several kits to purify our PCR products but none of them worked. Thus, we decided to directly continue with the digestion after the PCR without purifying. All the enzymes will be removed during the phenol/chloroform extraction just before the SELEX.

09.06.19

Bacteria culture - E. faecium and S. aureus

Few colonies of E. faecium and S. aureus were taken from the Petri dish culture of the 28.08 and grow in their respective liquid media at 37°C under agitation. After 3 hours of growth, they are centrifuged and the pellet of bacteria is resuspended in PBS 1X. Dilutions were performed until the OD reached between 0.5 and 0.6.

Phenol/chloroform extraction with Isopropanol - Digested products EFr1 & SAr1

The samples were purified after digestion. Then, they were analysed with a Nanodrop 2000 spectrophotometer.

Sample
Concetration (ng/μL)
260/280 ratio
260/230 ratio

EFr1 sample 1
17.2
1.46
1.29

EFr1 sample 2
26.5
1.49
1.44

EFr1 sample 3
29.8
1.49
1.47

EFr1 sample 4
37.2
1.56
1.5

SAr1 sample 1
69
1.51
1.54

SAr1 sample 2
61.5
1.75
0.28

Interpretations

A total quantity of 1728 pmol was obtained for E. faecium in the Round 1, and 869 pmol for S. aureus.

SELEX Round 2

The first round of SELEX was performed with 2 hours of incubation. The samples are then analyzed using a Nanodrop 2000 spectrophotometer.

Sample
Concetration (ng/μL)
260/280 ratio
260/230 ratio

SAr2
33.8
2.19
1.45

EFr2
32
2.21
1.6

PCR 10 - Amplification of EFr2 and SAr2

This PCR was performed to amplified the DNA after the second round of SELEX. The PCR followed the same program as described previously. It was run overnight and hold at 4°C.

Components
SAr1
EFr1

Phusion buffer
200
200

dNTPs
20
20

Forward primer
40
40

Reverse primer
40
40

Template DNA (1/3)
20
20

Phusion DNA polymerase
10
10

Nuclease-free water
670
670

Total volume
1000
1000

The samples are analyzed using a Nanodrop 2000 spectrophotometer.

Sample
Concetration (ng/μL)
260/280 ratio
260/230 ratio

PCR 10 - Round 2 - SA
1086.6
1.67
1.20

PCR 10 - Round 2 - EF
1243.4
1.66
1.20

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-           setTimeout(function(){window.scrollTo(0,(document.getElementById("PAGEOC5").offsetTop+document.getElementById("main").offsetTop)-50)},50);
+           setTimeout(function(){window.scrollTo(0,(document.getElementById("PAGEOC5").offsetTop+document.getElementById("main").offsetTop)-25)},50);
            qui =20;
          };
@@ Line 612: / Line 698: @@
          }
        </script>
-      <div id="PAGEJU1" class="pages">
+<div id="PAGEJU1" class="pages">
          <div class="NCroix" id="croixJU1">
-           <div style="height:10%;width:80%;background-color:white;position:relative;top:-40%;transform-origin:50% 50%;transform:rotate(45deg);left:10%;border-radius:3px 3px;float:left;">
+           <div style="height:10%;width:80%;background-color:white;position:relative;top:-40%;transform-origin:50% 50%;transform:rotate(45deg);left:10%;border-radius:3px 3px;float:left;"></div>
-          </div>
+           <div style="height:10%;width:80%;background-color:white;position:relative;top:-84%;transform-origin:50% 50%;transform:rotate(-45deg);left:10%;border-radius:3px 3px;float:left;"></div>
-           <div style="height:10%;width:80%;background-color:white;position:relative;top:-84%;transform-origin:50% 50%;transform:rotate(-45deg);left:10%;border-radius:3px 3px;float:left;">
-          </div>
          </div>
-         <div id="JU1" class="ent" style="overflow-x:hidden;overflow:scroll;width:85%;margin-left:7.5%;opacity:0;transition:0.3s;height:90%;">
+         <div id="JU1" class="ent">
            <p style="color:black;font-family:roboto;font-size:20px;font-weight:100;text-align:justify;margin-right:5%;">
-             Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
+             Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent purus augue,
-            purus augue, dictum non molestie eget, posuere a mauris. Donec
+            dictum non molestie eget, posuere a mauris. Donec
              fringilla eros in ipsum molestie eleifend. Maecenas viverra ex sed
              dolor blandit, at finibus orci rhoncus. Aliquam non dui est. In
@@ Line 725: / Line 809: @@
            </div>
          </div>
-         <div id="JU2" class="ent" style="overflow-x:hidden;overflow:scroll;width:85%;margin-left:7.5%;opacity:0;transition:0.3s;">
+         <div id="JU2" class="ent"  >
             <p style="color:black;font-family:roboto;font-size:20px;font-weight:100;text-align:justify;margin-right:5%;">
              Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
@@ Line 831: / Line 915: @@
            </div>
          </div>
-         <div id="JU3" class="ent" style="overflow-x:hidden;overflow:scroll;width:85%;margin-left:7.5%;opacity:0;transition:0.3s;">
+         <div id="JU3" class="ent"  >
          	 <p style="color:black;font-family:roboto;font-size:20px;font-weight:100;text-align:justify;margin-right:5%;">
              Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
@@ Line 937: / Line 1,021: @@
            </div>
          </div>
-         <div id="JU4" class="ent" style="overflow-x:hidden;overflow:scroll;width:85%;margin-left:7.5%;opacity:0;transition:0.3s;">
+         <div id="JU4" class="ent"  >
-           <p style="color:black;font-family:roboto;font-size:20px;font-weight:100;text-align:justify;margin-right:5%;">
+             <p class="p1N" style="z-index:2"> July Week 4 &nbsp; : &nbsp; 07/22 &nbsp; to &nbsp; 07/28 </p>
-             Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
+        <br><br><br><br>
-             purus augue, dictum non molestie eget, posuere a mauris. Donec
+        <p class="date">07.22.19</p>
-             fringilla eros in ipsum molestie eleifend. Maecenas viverra ex sed
+        <p class="p3N">Resuspension of oligonucleotides &nbsp;: </p>
-             dolor blandit, at finibus orci rhoncus. Aliquam non dui est. In
+        <p class="p2N">DNA library and primers were produced by Eurofins Genomics. Once received, they were resuspended according to the manufacturer’s instructions. Stock solutions of 100 µM were made by diluting the lyophilized oligonucleotides with a 10 mM Tris-EDTA pH 8.0 solution. Concentrations were checked using a Nanodrop 2000 spectrophotometer. </p><br>
-             mattis tincidunt nisl, at egestas felis dignissim id. Nam
+        <table class="notTABLE">
-             condimentum accumsan tellus quis ornare. Vestibulum quis augue
-             malesuada, tempor lorem ut, accumsan purus. Integer nibh nulla,
+            <tr class="NotTR">
-             malesuada ut sodales non, blandit non ligula. Donec venenatis
+              <td class="notTD">Samples</td>
-             faucibus pulvinar. Sed tincidunt ultrices eros, nec cursus dui
+              <td class="notTD">Concentration (ng/µl)</td>
-             molestie nec. Integer ac molestie lacus. Suspendisse lacinia mauris
+              <td class="notTD">260/280 ratio</td>
-             in dolor ullamcorper sodales. Lorem ipsum dolor sit amet,
+              <td class="notTD">260/230 ratio</td>
-             consectetur adipiscing elit. Pellentesque ut elit vitae neque luctus
+            </tr>
-             pellentesque porta sit amet orci.
+            <tr class="notTR">
-             Sed eget ante ipsum. Morbi pharetra lacinia purus, vitae efficitur
+              <td class="notTD">DNA library</td>
-             eros convallis vitae. Aenean in consequat tortor, vitae dapibus
+              <td class="notTD">2217.65</td>
-             nisi. Nunc non imperdiet tortor. Curabitur aliquet facilisis tempus.
+              <td class="notTD">1.89</td>
-             Integer viverra orci sit amet erat fringilla, sed interdum diam
+              <td class="notTD">1.56</td>
-             euismod. Ut ac vulputate orci. Nunc iaculis tellus quis massa
+            </tr>
-             convallis semper. Nunc nec consectetur arcu. In lectus lectus,
+            <tr class="notTR">
-             tempor id scelerisque vitae, egestas at purus. Curabitur vel erat
+              <td class="notTD">Reverse primer</td>
-             quis dui pretium lobortis nec a tellus.
+              <td class="notTD">8810.9</td>
-             Duis at efficitur sem. Suspendisse potenti. Donec pretium egestas
+              <td class="notTD">1.95</td>
-             suscipit. Vivamus in vulputate urna. Etiam ullamcorper aliquam
+              <td class="notTD">1.58</td>
-             blandit. Phasellus accumsan volutpat tortor non dignissim. Nunc
+            </tr>
-             vestibulum tellus vel ex pulvinar convallis. Etiam ultricies
-             convallis malesuada. Praesent sed cursus lorem, vitae iaculis ipsum.
+        </table>
-             Vestibulum risus risus, vulputate non metus non, posuere finibus
+        <br>
-             magna. Nulla hendrerit lorem ac lacus ornare porta. Vivamus ligula
+        <p class="p3N"> PCR 1 &nbsp;-&nbsp; Amplification of the DNA library </p>
-             lorem, ullamcorper eu nisl eget, bibendum condimentum dui.
+        <p class="p2N"> In order to start the SELEX, we need to amplify our DNA library in order to get enough material to start with. We tried several PCR protocols to optimize the amplification. This test was performed in order to know if the magnesium concentration could impact the quality of the amplification, and different annealing temperatures from 44 to 54°C were tested. A mix of Taq DNA polymerase and phusion DNA polymerase was used. </p>
-             Aenean consectetur tincidunt ullamcorper. Vestibulum tempus, mi nec
+        <table class="notTABLE" style="border-color:white;">
-             gravida euismod, massa est consectetur ligula, vitae mattis nunc
+          <tr class="notTR">
-             mauris vel tortor. Vivamus ac mauris ac odio mollis volutpat vitae
+            <td></td>
-             ac felis. Proin eu ex et erat commodo vulputate. Morbi quis velit
+            <td></td>
-             eleifend libero finibus hendrerit. Mauris egestas ultrices odio in
+            <td colspan="3" class="notTD" >Volume needed (&mu;L)</td>
-             rutrum. Pellentesque ut laoreet purus. In vitae orci ac tellus
+            <td></td>
-             eleifend condimentum. Phasellus at purus quis orci tempor convallis
+            <td></td>
-             vitae non nibh.
+          </tr>
-             Cras hendrerit interdum lectus vel dictum. Donec nec lectus mattis,
+          <tr class="NotTR">
-             aliquam tortor vitae, convallis sem. Suspendisse vitae dui tortor.
+          	<td class="notTD">Components</td>
-             Lorem ipsum dolor sit amet, consectetur adipiscing elit. Suspendisse
+            <td class="notTD">Final concentration</td>
-             egestas, lacus a lacinia molestie, leo nibh malesuada velit, sed
+            <td class="notTD" style="width:11%">Mix 1</td>
-             iaculis nulla libero nec dui. Maecenas fringilla, massa ut pretium
+            <td class="notTD" style="width:11%">Mix 2</td>
-            hendrerit, lorem urna tristique ligula, sed pharetra eros diam vel
+            <td class="notTD" style="width:11%">Mix 3</td>
-             odio. Aenean consectetur eu massa id volutpat. Nunc vel ex laoreet,
+          </tr>
-             aliquam augue ac, ullamcorper nunc. Pellentesque ut tempor dolor, ac
+          <tr class="notTR">
-             eleifend ante.
+            <td class="notTD">Phusion HF buffer 5X</td>
-             Pellentesque accumsan mauris in ante venenatis, nec elementum enim
+            <td class="notTD">1X</td>
-             ornare. In varius rutrum odio imperdiet ultricies. Pellentesque nec
+            <td class="notTD">50</td>
-             feugiat tortor. Donec euismod leo orci, quis sodales felis placerat
+            <td class="notTD">50</td>
-             ac. Donec a nibh dapibus, vulputate elit non, pretium nisl. Sed at
+            <td class="notTD">50</td>
-             est massa. Fusce at semper tortor. In cursus lorem lectus, nec
+          </tr>
-             suscipit nibh euismod eu. Morbi malesuada, lacus ut pellentesque
+          <tr class="notTR">
-             semper, diam tellus vulputate lectus, et varius ligula velit vitae
+            <td class="notTD">25 mM dNTPs</td>
-             elit. Praesent gravida, ex sit amet ornare bibendum, metus turpis
+            <td class="notTD">0.20&nbsp; mM</td>
-             maximus ipsum, eget interdum elit urna eu quam. Fusce tempor eu elit
+            <td class="notTD">2</td>
-             id efficitur. Curabitur vitae lorem vel arcu posuere blandit at et
+             <td class="notTD">2</td>
-             dolor. Nunc posuere pharetra odio vitae iaculis. In blandit dolor
+            <td class="notTD">2</td>
-             quis magna cursus aliquet. Aliquam luctus hendrerit mauris ut
+          </tr>
-             tristique. Vivamus pharetra scelerisque nisl vel consequat.
+          <tr class="notTR">
-             Integer iaculis enim et dui cursus lacinia. Fusce nec ultricies
+            <td class="notTD">Forward primer</td>
-             quam, quis molestie augue. Cras mauris quam, eleifend quis sagittis
+            <td class="notTD">0.5&nbsp; &mu;M</td>
-             eu, venenatis non mauris. Nulla sollicitudin ut nisl at ultrices.
+             <td class="notTD">9.75</td>
-             Vivamus fermentum placerat purus et gravida. Integer vulputate quam
+             <td class="notTD">9.75</td>
-             nec nunc porttitor, vel gravida est aliquam. Aenean elementum eget
+             <td class="notTD">9.75</td>
-             velit ut tincidunt. Fusce sollicitudin, magna vitae sodales
+          </tr>
-             tincidunt, diam turpis fermentum urna, eget maximus est libero
+          <tr class="notTR">
-             lacinia nibh. Donec sagittis elementum molestie. Donec suscipit
+            <td class="notTD">Reverse primer</td>
-             maximus laoreet. Proin sed convallis enim. Nullam dignissim pharetra
+            <td class="notTD">0.5&nbsp; &mu;M</td>
-             dapibus. Aenean vel varius ipsum.
+             <td class="notTD">10.1</td>
-             Ut eget rutrum nunc, sit amet tempor arcu. Etiam a tempus magna.
+             <td class="notTD">10.1</td>
-            Vivamus leo nunc, consectetur non sagittis euismod, eleifend in
+             <td class="notTD">10.1</td>
-             ante. Etiam imperdiet at dui vitae lobortis. Donec at mattis ipsum.
+          </tr>
-             Nam ut ante condimentum, sodales mauris vel, tempor massa. Curabitur
+          <tr class="notTR">
-            feugiat libero consectetur velit viverra, nec fermentum leo
+             <td class="notTD">25 mM Magnesium</td>
-            ultrices. Nulla laoreet in orci consequat condimentum. Donec et
+            <td class="notTD">2.5 &nbsp;mM</td>
-            posuere mauris, sagittis eleifend augue.
+             <td class="notTD">25</td>
-            Nunc venenatis enim in auctor rutrum. Nullam molestie arcu quis
+            <td class="notTD">50</td>
-            turpis mollis mattis. Phasellus elementum lectus vitae odio
+            <td class="notTD">75</td>
-            tincidunt, vitae congue nisl volutpat. Fusce sodales fringilla neque
+          </tr>
-            sed posuere. Morbi risus sem, imperdiet iaculis neque ac, malesuada
+           <tr class="notTR">
-            porta lectus. Quisque congue vel lorem vel commodo. In sit amet
+            <td class="notTD">DNA library</td>
-            gravida purus. Aliquam erat volutpat.
+            <td class="notTD">10 &nbsp;ng</td>
-            Praesent pellentesque pharetra sem sed condimentum. Quisque a
+            <td class="notTD">22.5</td>
-            bibendum turpis, et malesuada ipsum. Nunc viverra quam in magna
+             <td class="notTD">22.5</td>
-            viverra facilisis. Ut pharetra eget urna sit amet luctus. Phasellus
+            <td class="notTD">22.5</td>
-            condimentum fermentum tortor, ut venenatis augue suscipit non.
+          </tr>
-            Maecenas facilisis arcu non diam vehicula lobortis. Suspendisse ut
+          <tr class="notTR">
-            neque libero. Quisque sagittis iaculis faucibus. Nam sagittis,
+             <td class="notTD">Taq DNA polymerase</td>
-            mauris ut vestibulum tincidunt, dui ante sagittis ex, id finibus
+            <td class="notTD">0.1&nbsp; U/&mu;L</td>
-             eros augue eu velit. Maecenas vulputate ante sed hendrerit
+             <td class="notTD">0.02</td>
-             vestibulum. Lorem ipsum dolor sit amet, consectetur adipiscing elit.
+             <td class="notTD">0.02</td>
-             Nam enim purus, porta in leo id, accumsan gravida augue. Vestibulum
+             <td class="notTD">0.02</td>
-            quis sollicitudin lectus, a pretium erat. Donec hendrerit imperdiet
+          </tr>
-            turpis, vel convallis est pharetra quis. Curabitur molestie
+          <tr class="notTR">
-            porttitor turpis, in fringilla arcu lacinia et. </p>
+             <td class="notTD">Phusion DNA polymerase</td>
+            <td class="notTD">1&nbsp; &nbsp; U/&mu;L</td>
+            <td class="notTD">0.5</td>
+             <td class="notTD">0.5</td>
+            <td class="notTD">0.5</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Nuclease-free water</td>
+             <td class="notTD">/</td>
+            <td class="notTD">130.13</td>
+             <td class="notTD">105.13</td>
+            <td class="notTD">80.13</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Total volume</td>
+            <td class="notTD">/</td>
+            <td class="notTD">250</td>
+            <td class="notTD">250</td>
+            <td class="notTD">250</td>
+          </tr>
+        </table>
+        <br>
+        <p class="p2N">
+          <i>Rq: Enzymes were added at the end as hot-start PCR protocols can optimize the yield of the desired product while limiting the likelihood of nonspecific amplification</i>
+        </p>
+        <br>
+        <p class="p3N">
+          PCR plate plan
+        </p>
+        <table class="notTABLE">
+          <tr class="notTR" style="background-color:#bcbcbc">
+            <td class="notTD" style="width:25%">A (54°C)</td>
+            <td class="notTD" style="width:25%" >1.1</td>
+             <td class="notTD" style="width:25%">2.1</td>
+             <td class="notTD" style="width:25%">3.1</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD" >B</td>
+            <td class="notTD" >/</td>
+            <td class="notTD" >/</td>
+            <td class="notTD" >/</td>
+          </tr>
+          <tr class="notTR" style="background-color:#bcbcbc">
+            <td class="notTD" >C (51.9°C)</td>
+             <td class="notTD" >1.2</td>
+             <td class="notTD" >2.2</td>
+             <td class="notTD" >3.2</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD" >D</td>
+             <td class="notTD" >/</td>
+            <td class="notTD" >/</td>
+            <td class="notTD" >/</td>
+          </tr>
+          <tr class="notTR" style="background-color:#bcbcbc">
+            <td class="notTD" >E (47.6°C)</td>
+            <td class="notTD" >1.3</td>
+             <td class="notTD" >2.3</td>
+             <td class="notTD" >3.3</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD" >F</td>
+             <td class="notTD" >/</td>
+            <td class="notTD" >/</td>
+            <td class="notTD" >/</td>
+          </tr>
+          <tr class="notTR" style="background-color:#bcbcbc">
+            <td class="notTD" >G (44.7°C)</td>
+             <td class="notTD" >1.4</td>
+            <td class="notTD" >2.4</td>
+             <td class="notTD" >3.4</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD" >G</td>
+            <td class="notTD" >/</td>
+            <td class="notTD" >/</td>
+            <td class="notTD" >/</td>
+          </tr>
+        </table>
+        <br><br>
+        <p class="p3N">
+          PCR program used:</p>
+        <br>
+        <p class="p2N" style="text-indent:0px;line-height:0em;">
+          - &nbsp;&nbsp;&nbsp;95°C &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5 min<br></p>
+          <p class="p2N" style="text-indent:0px;font-weight:400;line-height:0em;">
+          - &nbsp;&nbsp;&nbsp;95°C &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;30 s</p>
+        <p class="p2N" style="text-indent:0px;font-weight:400;line-height:0em;">
+          - &nbsp;&nbsp;&nbsp;44-54°C &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1 min  &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;30 cycles</p>
+        <p class="p2N" style="text-indent:0px;font-weight:400;line-height:0em;">
+          - &nbsp;&nbsp;&nbsp;68°C &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;30 s&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</p>
+        <p class="p2N" style="text-indent:0px;line-height:0em;">
+          - &nbsp;&nbsp;&nbsp;68°C &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5 min</p>
+          <p class="p2N" style="text-indent:0px;line-height:0em;">
+          - &nbsp;&nbsp;&nbsp;15°C &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HOLD ALL NIGHT
+        </p>
+        <br>
+        <p class="p2N" style="text-indent:0px">
+          PCR was run overnight. Samples were checked using a Nanodrop 2000 spectrophotometer.
+        </p>
+        <br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+             <td class="notTD" style="width:25%;">Sample</td>
+            <td class="notTD" style="width:25%;">Concentration (ng/&mu;L)</td>
+            <td class="notTD" style="width:25%;">260/280 ratio</td>
+            <td class="notTD" style="width:25%;">260/230 ratio</td>
+          </tr>
+          <tr class="notTR">
+          	<td class="notTD">1.1</td>
+             <td class="notTD">465.9</td>
+             <td class="notTD">1.39</td>
+             <td class="notTD">0.66</td>
+          </tr>
+           <tr class="notTR">
+          	<td class="notTD">1.2</td>
+             <td class="notTD">501.7</td>
+             <td class="notTD">1.42</td>
+             <td class="notTD">0.69</td>
+          </tr>
+          <tr class="notTR">
+          	<td class="notTD">1.3</td>
+             <td class="notTD">480.4</td>
+            <td class="notTD">1.43</td>
+             <td class="notTD">0.68</td>
+          </tr>
+          <tr class="notTR">
+          	<td class="notTD">1.4</td>
+             <td class="notTD">469.9</td>
+             <td class="notTD">1.43</td>
+             <td class="notTD">0.65</td>
+          </tr>
+          <tr class="notTR" style="background-color:#bcbcbc">
+          	<td class="notTD">2.1</td>
+             <td class="notTD">498.3</td>
+             <td class="notTD">1.40</td>
+             <td class="notTD">0.68</td>
+          </tr>
+          <tr class="notTR" style="background-color:#bcbcbc">
+          	<td class="notTD">2.2</td>
+             <td class="notTD">498.7</td>
+             <td class="notTD">1.43</td>
+             <td class="notTD">0.70</td>
+          </tr>
+          <tr class="notTR" style="background-color:#bcbcbc">
+          	<td class="notTD">2.3</td>
+             <td class="notTD">532.9</td>
+             <td class="notTD">1.42</td>
+             <td class="notTD">0.71</td>
+          </tr>
+          <tr class="notTR" style="background-color:#bcbcbc">
+          	<td class="notTD">2.4</td>
+             <td class="notTD">495.2</td>
+             <td class="notTD">1.42</td>
+            <td class="notTD">0.69</td>
+          </tr>
+          <tr class="notTR">
+          	<td class="notTD">3.1</td>
+             <td class="notTD">497.8</td>
+             <td class="notTD">1.43</td>
+             <td class="notTD">0.70</td>
+          </tr>
+          <tr class="notTR">
+          	<td class="notTD">3.2</td>
+             <td class="notTD">512.2</td>
+             <td class="notTD">1.43</td>
+             <td class="notTD">0.70</td>
+          </tr>
+          <tr class="notTR">
+          	<td class="notTD">3.3</td>
+             <td class="notTD">505.5</td>
+             <td class="notTD">1.42</td>
+             <td class="notTD">0.70</td>
+          </tr>
+          <tr class="notTR">
+          	<td class="notTD">3.4</td>
+             <td class="notTD">527.2</td>
+            <td class="notTD">1.41</td>
+             <td class="notTD">0.70</td>
+          </tr>
+        </table>
+        <br>
+        <p class="p3N">
+          Interpretations :
+        </p>
+        <p class="p2N" style="text-indent:0px">
+          No special condition seemed better than others.
+        </p>
+        <br><br><br><br>
+        <p class="date">
+.23.19
+        </p>
+        <p class="p3N">
+          Gel electrophoresis 1 &nbsp;-&nbsp; Amplified library
+        </p><br>
+        <p class="p2N">
+          To verify that the samples were all the same with the right size, we performed a gel electrophoresis using a 3% agarose gel. Each band was cut and then kept at -4°C in order to be purified later.
+        </p>
+        <br>
+        <div style="height:67vh;">
+          <div style="float:left;width:40%;">
+             <img src="https://2019.igem.org/wiki/images/9/94/T--Pasteur_Paris--NotbookImg1.jpg" width="100%">
+          </div>
+          <div style="float:left;width:50%;margin-left:8%;margin-top:1.5vw;">
+             <table class="notTABLE">
+              <tr class="notTR">
+                <td style="text-align:center;width:20%;font-weight:400;padding:10px;">1.</td>
+                <td>Sample 1.1 (54°C, 25 &nbsp;&mu;L, Mg2+)</td>
+              </tr>
+              <tr class="notTR">
+                <td style="text-align:center;width:20%;font-weight:400;padding:10px;">2.</td>
+                <td>&nbsp;&nbsp;&nbsp;Sample 1.2 (51.9°C, 25 &nbsp;&mu;L, Mg2+)</td>
+              </tr>
+              <tr class="notTR">
+                <td style="text-align:center;width:20%;font-weight:400;padding:10px;">3.</td>
+                <td>&nbsp;&nbsp;&nbsp;Sample 1.3 (47.6°C, 25 &nbsp;&mu;L, Mg2+)</td>
+              </tr>
+              <tr class="notTR">
+                <td style="text-align:center;width:20%;font-weight:400;padding:10px;">4.</td>
+                <td>&nbsp;&nbsp;&nbsp;Sample 1.4 (44.7°C, 25 &nbsp;&mu;L, Mg2+)</td>
+              </tr>
+               <tr class="notTR">
+                <td style="text-align:center;width:20%;font-weight:400;padding:10px;">5.</td>
+                <td>Sample 2.1 (54°C, 50 &nbsp;&mu;L, Mg2+)</td>
+              </tr>
+              <tr class="notTR">
+                <td style="text-align:center;width:20%;font-weight:400;padding:10px;">6.</td>
+                <td>&nbsp;&nbsp;&nbsp;Sample 2.2 (51.9°C, 50 &nbsp;&mu;L, Mg2+)</td>
+              </tr>
+              <tr class="notTR">
+                <td style="text-align:center;width:20%;font-weight:400;padding:10px;">7.</td>
+                <td>&nbsp;&nbsp;&nbsp;Sample 2.3 (47.6°C, 50 &nbsp;&mu;L, Mg2+)</td>
+              </tr>
+              <tr class="notTR">
+                <td style="text-align:center;width:20%;font-weight:400;padding:10px;">8.</td>
+                <td>&nbsp;&nbsp;&nbsp;Sample 2.4 (44.7°C, 50 &nbsp;&mu;L, Mg2+)</td>
+              </tr>
+              <tr class="notTR">
+                <td style="text-align:center;width:20%;font-weight:400;padding:10px;">9.</td>
+                <td>Sample 3.1 (54°C, 75 &nbsp;&mu;L, Mg2+)</td>
+              </tr>
+              <tr class="notTR">
+                <td style="text-align:center;width:20%;font-weight:400;padding:10px;">10.</td>
+                <td>&nbsp;&nbsp;&nbsp;Sample 3.2 (51.9°C, 75 &nbsp;&mu;L, Mg2+)</td>
+              </tr>
+              <tr class="notTR">
+                <td style="text-align:center;width:20%;font-weight:400;padding:10px;">11.</td>
+                <td>&nbsp;&nbsp;&nbsp;Sample 3.3 (47.6°C, 75 &nbsp;&mu;L, Mg2+)</td>
+              </tr>
+              <tr class="notTR">
+                <td style="text-align:center;width:20%;font-weight:400;padding:10px;">12.</td>
+                <td>&nbsp;&nbsp;&nbsp;Sample 3.4 (44.7°C, 75 &nbsp;&mu;L, Mg2+)</td>
+              </tr>
+             </table>
+          </div>
+          <br><br>
+        </div>
+         <p class="p3N" style="margin-top:50px">
+             Interpretations
+          </p>
+          <p class="p2N">
+            All samples had the same size of 120 bp approximately whereas the library size was 80 bp normally.
+          </p>
+          <br><br>
+          <p class="p3N">
+             Conclusion
+          </p>
+          <p class="p2N">
+            PCR amplification resulted in a shift in size of the oligonucleotides because of the double-stranded form.
+          </p>
+        <br>
+        <br>
+        <p class="p3N">
+          Cell culture
+        </p>
+        <p class="p2N">
+          TO COMPLETE
+        </p>
+        <p class="date">
+.24.19
+        </p>
+        <p class="p3N">
+          Growth curves
+        </p>
+        <br>
+        <p class="p2N">
+          Bacteria were cultured and their growth was analyzed by establishing a growth curve associated with the number of colonies. Several strains were used:
+        </p>
+        <p class="p2N">
+          <li style="font-family:roboto;font-size:20px;font-weight:100;text-indent:50px;line-height:1.5em;">E. Faecium (ref CIP 103014T)</li>
+          <li style="font-family:roboto;font-size:20px;font-weight:100;text-indent:50px;line-height:1.5em;">P. Aeruginosa (ref 104116)</li>
+          <li style="font-family:roboto;font-size:20px;font-weight:100;text-indent:50px;line-height:1.5em;">A. Baumanii (ref CIP 79.34T)</li>
+          <li style="font-family:roboto;font-size:20px;font-weight:100;text-indent:50px;line-height:1.5em;">S. Aureus (ref CIP 103429)</li>
+          <li style="font-family:roboto;font-size:20px;font-weight:100;text-indent:50px;line-height:1.5em;">S. Pneumoniae (ref CIP 104340)</li>
+          <li style="font-family:roboto;font-size:20px;font-weight:100;text-indent:50px;line-height:1.5em;">E. Coli (ref 53.126)</li>
+          <li style="font-family:roboto;font-size:20px;font-weight:100;text-indent:50px;line-height:1.5em;">E. Enterica (ref CIP 60.62T)</li>
+          <br><br>
+        </p>
+        <p class="p2N">
+          All strains were provided by the CRBIP of the Pasteur Institute.
+        </p>
+        <p class="p2N">
+          TO COMPLETE
+        </p>
+        <p class="date">
+.25.19
+        </p>
+        <p class="p3N">
+          Purification of PCR products &nbsp;-&nbsp; Invitrogen Pure Link Quick Gel extraction kit &nbsp;-&nbsp; PCR 1
+        </p>
+        <p class="p2N">
+          After PCR amplification, PCR products need to be cleaned up. Gel slices were extracted and purified using Invitrogen Pure Link Quick Gel extraction kit following the manufacturer’s instructions.
+        </p>
+        <div >
+          <img src="https://2019.igem.org/wiki/images/b/b2/T--Pasteur_Paris--NotebookImg2.png" style="width:70%;margin-left:10%">
          </div>
+        <br><br><br><br>
+        <p class="p3N">
+          END OF THE WEEK
+        </p>
+      </div>
        </div>
        <div id="PAGEJU5" class="pages">
@@ Line 1,043: / Line 1,447: @@
            </div>
          </div>
-         <div id="JU5" class="ent" style="overflow-x:hidden;overflow:scroll;width:85%;margin-left:7.5%;opacity:0;transition:0.3s;">
+         <div id="JU5" class="ent"  >
-           <p style="color:black;font-family:roboto;font-size:20px;font-weight:100;text-align:justify;margin-right:5%;">
+           <p class="p1N" style="z-index:2"> July Week 5 &nbsp; : &nbsp; 07/29
-             Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
+          &nbsp; to &nbsp; 08/04 </p>
-             purus augue, dictum non molestie eget, posuere a mauris. Donec
+        <br><br><br><br>
-             fringilla eros in ipsum molestie eleifend. Maecenas viverra ex sed
+        <p class="date"> 07.29.19</p>
-             dolor blandit, at finibus orci rhoncus. Aliquam non dui est. In
+        <p class="p3N"> Gel electrophoresis 2 &nbsp;-&nbsp; Purified PCR products from gel extraction kit </p><br>
-             mattis tincidunt nisl, at egestas felis dignissim id. Nam
+        <p class="p2N">To verify that the samples were purified after gel extraction, we performed a gel electrophoresis using a 3% agarose gel. We used two ladders, one at 25 bp and the other at 50 bp from Promega.
-             condimentum accumsan tellus quis ornare. Vestibulum quis augue
+        </p><br>
-             malesuada, tempor lorem ut, accumsan purus. Integer nibh nulla,
+        <div>
-             malesuada ut sodales non, blandit non ligula. Donec venenatis
+          <img src="https://2019.igem.org/wiki/images/5/54/T--Pasteur_Paris--NotebookImg3.png" style="width:70%;margin-left:7%;">
-             faucibus pulvinar. Sed tincidunt ultrices eros, nec cursus dui
+        </div>
-             molestie nec. Integer ac molestie lacus. Suspendisse lacinia mauris
+        <br><br>
-             in dolor ullamcorper sodales. Lorem ipsum dolor sit amet,
+        <p class="p3N">
-             consectetur adipiscing elit. Pellentesque ut elit vitae neque luctus
+          Interpretation
-             pellentesque porta sit amet orci.
+        </p>
-             Sed eget ante ipsum. Morbi pharetra lacinia purus, vitae efficitur
+        <p class="p2N">
-             eros convallis vitae. Aenean in consequat tortor, vitae dapibus
+          There was no band after purification by gel extraction. So, during the purification step we lost the DNA.
-             nisi. Nunc non imperdiet tortor. Curabitur aliquet facilisis tempus.
+        </p>
-             Integer viverra orci sit amet erat fringilla, sed interdum diam
+        <br><br>
-             euismod. Ut ac vulputate orci. Nunc iaculis tellus quis massa
+        <p class="p3N">
-             convallis semper. Nunc nec consectetur arcu. In lectus lectus,
+          Digestion &nbsp;-&nbsp; Amplified library
-             tempor id scelerisque vitae, egestas at purus. Curabitur vel erat
+        </p>
-             quis dui pretium lobortis nec a tellus.
+        <p class="p2N">
-             Duis at efficitur sem. Suspendisse potenti. Donec pretium egestas
+          This step is made to pass from a double-stranded DNA to a single-stranded one.
-             suscipit. Vivamus in vulputate urna. Etiam ullamcorper aliquam
+We performed the digestion using Lambda Exonuclease from New England Biolabs directly on the amplified library (PCR 1) without purifying it beforehand.
-             blandit. Phasellus accumsan volutpat tortor non dignissim. Nunc
+        </p>
-             vestibulum tellus vel ex pulvinar convallis. Etiam ultricies
+        <p class="p2N" style="text-indent:100px">
-             convallis malesuada. Praesent sed cursus lorem, vitae iaculis ipsum.
+. &nbsp;&nbsp;Set-up the reaction as follows:
-             Vestibulum risus risus, vulputate non metus non, posuere finibus
+        </p>
-             magna. Nulla hendrerit lorem ac lacus ornare porta. Vivamus ligula
-             lorem, ullamcorper eu nisl eget, bibendum condimentum dui.
+        <table class="notTABLE">
-             Aenean consectetur tincidunt ullamcorper. Vestibulum tempus, mi nec
+          <tr class="NotTR">
-             gravida euismod, massa est consectetur ligula, vitae mattis nunc
+            <td class="notTD">Components</td>
-             mauris vel tortor. Vivamus ac mauris ac odio mollis volutpat vitae
+            <td class="notTD">Volume for 50 &nbsp;&mu;L reaction</td>
-             ac felis. Proin eu ex et erat commodo vulputate. Morbi quis velit
+          </tr>
-             eleifend libero finibus hendrerit. Mauris egestas ultrices odio in
+          <tr class="notTR">
-             rutrum. Pellentesque ut laoreet purus. In vitae orci ac tellus
+            <td class="notTD">DNA from PCR 1</td>
-             eleifend condimentum. Phasellus at purus quis orci tempor convallis
+            <td class="notTD">5 &nbsp;&mu;g</td>
-             vitae non nibh.
+          </tr>
-             Cras hendrerit interdum lectus vel dictum. Donec nec lectus mattis,
+          <tr class="notTR">
-             aliquam tortor vitae, convallis sem. Suspendisse vitae dui tortor.
+            <td class="notTD">Lambda Exonuclease Reaction Buffer (10X)</td>
-             Lorem ipsum dolor sit amet, consectetur adipiscing elit. Suspendisse
+            <td class="notTD">5&nbsp;&mu;L &nbsp;(1X)</td>
-             egestas, lacus a lacinia molestie, leo nibh malesuada velit, sed
+          </tr>
-             iaculis nulla libero nec dui. Maecenas fringilla, massa ut pretium
+          <tr class="notTR">
-             hendrerit, lorem urna tristique ligula, sed pharetra eros diam vel
+            <td class="notTD">Lambda Exonuclease</td>
-             odio. Aenean consectetur eu massa id volutpat. Nunc vel ex laoreet,
+            <td class="notTD">1 &mu;L &nbsp;(5 units)</td>
-             aliquam augue ac, ullamcorper nunc. Pellentesque ut tempor dolor, ac
+          </tr>
-             eleifend ante.
+          <tr class="notTR">
-             Pellentesque accumsan mauris in ante venenatis, nec elementum enim
+            <td class="notTD">Nuclease-free water</td>
-             ornare. In varius rutrum odio imperdiet ultricies. Pellentesque nec
+            <td class="notTD">up to 50&mu;L</td>
-             feugiat tortor. Donec euismod leo orci, quis sodales felis placerat
+          </tr>
-             ac. Donec a nibh dapibus, vulputate elit non, pretium nisl. Sed at
+        </table>
-             est massa. Fusce at semper tortor. In cursus lorem lectus, nec
+        <p class="p2N" style="text-indent:100px">
-             suscipit nibh euismod eu. Morbi malesuada, lacus ut pellentesque
+. &nbsp;&nbsp;Incubate at 37°C for 30 minutes.
-             semper, diam tellus vulputate lectus, et varius ligula velit vitae
+        </p>
-             elit. Praesent gravida, ex sit amet ornare bibendum, metus turpis
+        <p class="p2N" style="text-indent:100px">
-             maximus ipsum, eget interdum elit urna eu quam. Fusce tempor eu elit
+. &nbsp;&nbsp;Stop reaction by adding EDTA to 10 mM.
-             id efficitur. Curabitur vitae lorem vel arcu posuere blandit at et
+        </p>
-             dolor. Nunc posuere pharetra odio vitae iaculis. In blandit dolor
+         <p class="p2N" style="text-indent:100px">
-             quis magna cursus aliquet. Aliquam luctus hendrerit mauris ut
+. &nbsp;&nbsp;Heat at 75°C for 10 minutes.
-             tristique. Vivamus pharetra scelerisque nisl vel consequat.
+        </p><br><br>
-             Integer iaculis enim et dui cursus lacinia. Fusce nec ultricies
+        <p class="date">
-             quam, quis molestie augue. Cras mauris quam, eleifend quis sagittis
+.31.19
-             eu, venenatis non mauris. Nulla sollicitudin ut nisl at ultrices.
+        </p>
-             Vivamus fermentum placerat purus et gravida. Integer vulputate quam
+        <p class="p3N">
-             nec nunc porttitor, vel gravida est aliquam. Aenean elementum eget
+          PCR 2 &nbsp;-&nbsp; Testing different conditions
-             velit ut tincidunt. Fusce sollicitudin, magna vitae sodales
+        </p>
-             tincidunt, diam turpis fermentum urna, eget maximus est libero
+        <br>
-             lacinia nibh. Donec sagittis elementum molestie. Donec suscipit
+        <p class="p2N">
-             maximus laoreet. Proin sed convallis enim. Nullam dignissim pharetra
+          To try to enhance our PCR protocol, we performed a new PCR using different conditions. These conditions enabled us to determine if the Taq DNA Polymerase has to be activated prior addition, and if both polymerases have to be added at 95°C or not. The PCR was run overnight and hold at 4°C.
-             dapibus. Aenean vel varius ipsum.
+        </p>
-             Ut eget rutrum nunc, sit amet tempor arcu. Etiam a tempus magna.
+        <table class="notTABLE" style="border-color:white">
-             Vivamus leo nunc, consectetur non sagittis euismod, eleifend in
+          <tr class="notTR">
-             ante. Etiam imperdiet at dui vitae lobortis. Donec at mattis ipsum.
+            <td></td>
-             Nam ut ante condimentum, sodales mauris vel, tempor massa. Curabitur
+            <td colspan="2" class="notTD">Volume needed (in &mu;L)</td>
-             feugiat libero consectetur velit viverra, nec fermentum leo
+            <td></td>
-             ultrices. Nulla laoreet in orci consequat condimentum. Donec et
+          </tr>
-             posuere mauris, sagittis eleifend augue.
+          <tr class="NotTR">
-             Nunc venenatis enim in auctor rutrum. Nullam molestie arcu quis
+            <td class="notTD" style="width:55%">Components</td>
-             turpis mollis mattis. Phasellus elementum lectus vitae odio
+						<td class="notTD">Mix 1</td>
-             tincidunt, vitae congue nisl volutpat. Fusce sodales fringilla neque
+            <td class="notTD">Mix 2</td>
-             sed posuere. Morbi risus sem, imperdiet iaculis neque ac, malesuada
+          </tr>
-             porta lectus. Quisque congue vel lorem vel commodo. In sit amet
+          <tr class="notTR">
-             gravida purus. Aliquam erat volutpat.
+            <td class="notTD" style="width:55%">Phusion HF buffer 5X</td>
-             Praesent pellentesque pharetra sem sed condimentum. Quisque a
+						<td class="notTD">500</td>
-             bibendum turpis, et malesuada ipsum. Nunc viverra quam in magna
+            <td class="notTD">500</td>
-             viverra facilisis. Ut pharetra eget urna sit amet luctus. Phasellus
+          </tr>
-             condimentum fermentum tortor, ut venenatis augue suscipit non.
+          <tr class="notTR">
-             Maecenas facilisis arcu non diam vehicula lobortis. Suspendisse ut
+            <td class="notTD" style="width:55%">25 mM dNTPs</td>
-             neque libero. Quisque sagittis iaculis faucibus. Nam sagittis,
+						<td class="notTD">20</td>
-            mauris ut vestibulum tincidunt, dui ante sagittis ex, id finibus
+            <td class="notTD">250</td>
-             eros augue eu velit. Maecenas vulputate ante sed hendrerit
+          </tr>
-             vestibulum. Lorem ipsum dolor sit amet, consectetur adipiscing elit.
+          <tr class="notTR">
-             Nam enim purus, porta in leo id, accumsan gravida augue. Vestibulum
+            <td class="notTD" style="width:55%">Forward primer</td>
-            quis sollicitudin lectus, a pretium erat. Donec hendrerit imperdiet
+						<td class="notTD">97.5</td>
-            turpis, vel convallis est pharetra quis. Curabitur molestie
+            <td class="notTD">50</td>
-            porttitor turpis, in fringilla arcu lacinia et. </p>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD" style="width:55%">Reverse primer</td>
+						<td class="notTD">101</td>
+            <td class="notTD">50</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD" style="width:55%">25 mM Magnesium</td>
+						<td class="notTD">250</td>
+            <td class="notTD">250</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD" style="width:55%">DNA library (1/1000 dilution)</td>
+						<td class="notTD">225</td>
+            <td class="notTD">250</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD" style="width:55%">Taq DNA polymerase diluted 1/10</td>
+						<td class="notTD">5</td>
+            <td class="notTD">5</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD" style="width:55%">Phusion DNA polymerase</td>
+						<td class="notTD">25</td>
+            <td class="notTD">25</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD" style="width:55%">Nuclease-free water</td>
+						<td class="notTD">1276.5</td>
+            <td class="notTD">1135</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD" style="width:55%">Total volume</td>
+						<td class="notTD">2500</td>
+            <td class="notTD">2500</td>
+          </tr>
+        </table>
+        <br>
+        <p class="p3N">
+          PCR plan
+        </p>
+        <table class="notTABLE" style="border-color:white;">
+          <tr class="notTR">
+             <td class="notTD" style="width:7.69%"></td>
+             <td class="notTD" style="width:7.69%">1</td>
+             <td class="notTD" style="width:7.69%">2</td>
+             <td class="notTD" style="width:7.69%">3</td>
+            <td class="notTD" style="width:7.69%">4</td>
+             <td class="notTD" style="width:7.69%">5</td>
+             <td class="notTD" style="width:7.69%">6</td>
+             <td class="notTD" style="width:7.69%">7</td>
+             <td class="notTD" style="width:7.69%">8</td>
+             <td class="notTD" style="width:7.69%">9</td>
+             <td class="notTD" style="width:7.69%">10</td>
+            <td class="notTD" style="width:7.69%">11</td>
+             <td class="notTD" style="width:7.69%">12</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD" style="width:7.69%;">A</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">1A</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">2A</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">3A</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">4A</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">5A</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">6A</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">7A</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">8A</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">9A</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">10A</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">11A</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">12A</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD" style="width:7.69%;">B</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">1B</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">2B</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">3B</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">4B</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">5B</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">6B</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">7B</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">8B</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">9B</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">10B</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">11B</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">12B</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD" style="width:7.69%;">C</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">1C</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">2C</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">3C</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">4C</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">5C</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">6C</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">7C</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">8C</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">9C</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">10C</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">11C</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">12C</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD" style="width:7.69%;">D</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">1D</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">2D</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">3D</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">4D</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">5D</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">6D</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">7D</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">8D</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">9D</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">10D</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">11D</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">12D</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD" style="width:7.69%;">E</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">1E</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">2E</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">3E</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">4E</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">5E</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">6E</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">7E</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">8E</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">9E</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">10E</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">11E</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">12E</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD" style="width:7.69%;">F</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">1F</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">2F</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">3F</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">4F</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">5F</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">6F</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">7F</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">8F</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">9F</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">10F</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">11F</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">12F</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD" style="width:7.69%;">G</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">1G</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">2G</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">3G</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">4G</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">5G</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">6G</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">7G</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">8G</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">9G</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">10G</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">11G</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">12G</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD" style="width:7.69%;">H</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">1H</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">2H</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">3H</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">4H</td>
+            <td class="notTD" style="width:7.69%;background-color:#9d9d9d">5H</td>
+             <td class="notTD" style="width:7.69%;background-color:#9d9d9d">6H</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">7H</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">8H</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">9H</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">10H</td>
+            <td class="notTD" style="width:7.69%;background-color:#bcbcbc">11H</td>
+             <td class="notTD" style="width:7.69%;background-color:#bcbcbc">12H</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD" style="width:7.69%;"></td>
+             <td colspan="6" class="notTD">Mix 1</td>
+            <td colspan="6"class="notTD">Mix 2</td>
+          </tr>
+        </table>
+        <br><br>
+        <p class="p3N">
+          Legend &nbsp;:
+        </p>
+        <p class="p2N">
+          <li style="font-family:roboto;font-size:20px;text-indent:100px;">Condition 1: Taq was activated and added with Phusion in ice</li>
+          <li style="font-family:roboto;font-size:20px;text-indent:100px;">Condition 2: Taq was not activated and added with Phusion in ice</li>
+          <li style="font-family:roboto;font-size:20px;text-indent:100px;">Condition 3: Phusion was added in ice</li>
+          <li style="font-family:roboto;font-size:20px;text-indent:100px;">Condition 4: Taq was activated and added with Phusion at 95°C</li>
+          <li style="font-family:roboto;font-size:20px;text-indent:100px;">Condition 5: Taq was not activated and added with Phusion at 95°C</li>
+          <li style="font-family:roboto;font-size:20px;text-indent:100px;">Condition 6: Phusion was added at 95°C</li>
+          <li style="font-family:roboto;font-size:20px;text-indent:100px;">Condition 7: Taq was activated and added with Phusion in ice</li>
+          <li style="font-family:roboto;font-size:20px;text-indent:100px;">Condition 8: Taq was not activated and added with Phusion in ice</li>
+          <li style="font-family:roboto;font-size:20px;text-indent:100px;">Condition 9: Phusion was added in ice</li>
+          <li style="font-family:roboto;font-size:20px;text-indent:100px;">Condition 10: Taq was activated and added with Phusion at 95°C</li>
+          <li style="font-family:roboto;font-size:20px;text-indent:100px;">Condition 11: Taq was not activated and added with Phusion at 95°C</li>
+          <li style="font-family:roboto;font-size:20px;text-indent:100px;">Condition 12: Phusion was added at 95°C</li>
+        </p>
+        <br>
+        <p class="p3N">
+          Gel electrophoresis 4 &nbsp;-&nbsp; PCR 2
+        </p>
+        <p class="p2N">
+          A gel containing 3% of agarose was run to determine which condition led to the best results.
+        </p>
+        <div style="margin-top:5%">
+          <img src="https://2019.igem.org/wiki/images/a/ad/T--Pasteur_Paris--NotebookImg4.png" style="width:80%;margin-left:5%;">
+        </div>
+        <br>
+        <br>
+        <p class="p3N">
+          Interpretation
+        </p>
+        <br>
+        <p class="p2N">
+          Condition 1 provided the best results because the band is the brightest. Moreover, there was almost no difference between the samples containing activated Taq polymerase and when the polymerases were added on ice or in 95°C. Because we obtained the same results when we amplified using both Taq and phusion polymerases and only Phusion polymerase, we decided to only use Phusion polymerase for our PCR.
+        </p>
+        <br><br>
+        <p class="date">
+.01.19
+        </p>
+        <p class="p3N">
+          DNA precipitation &nbsp;-&nbsp; Product of phenol/chloroform from 29.07.19
+        </p>
+        <p class="p2N" style="text-indent:50px">
+. &nbsp;&nbsp;Add 1/10 volume of Sodium Acetate (3 M, pH 5.2) and 2,5x volume of 100% ethanol
+        </p>
+        <table class="notTABLE">
+          <tr class="NotTR">
+             <td class="notTD" style="width:33%">Sample</td>
+            <td class="notTD">Pool 1 Elution 1</td>
+            <td class="notTD">Pool 1 Elution 2</td>
+            <td class="notTD">Pool 2 Elution 1</td>
+            <td class="notTD">Pool 2 Elution 2</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD" style="width:33%">Upper aqueous phase recovered</td>
+            <td class="notTD">2000 µL</td>
+            <td class="notTD">430 µL</td>
+            <td class="notTD">1150 µL</td>
+            <td class="notTD">415 µL</td>
+          </tr>
+           <tr class="notTR">
+            <td class="notTD" style="width:33%">Sodium acetate 3M to add</td>
+            <td class="notTD">200 µL</td>
+            <td class="notTD">43 µL</td>
+            <td class="notTD">115 µL</td>
+            <td class="notTD">41.5 µL</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD" style="width:33%">Volume after addition of sodium acetate</td>
+            <td class="notTD">2200 µL</td>
+            <td class="notTD">473 µL</td>
+            <td class="notTD">1265 µL</td>
+            <td class="notTD">456.5 µL</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD" style="width:33%">Ethanol 100%  to add</td>
+            <td class="notTD">5500 µL</td>
+            <td class="notTD">1182.5 µL</td>
+             <td class="notTD">3162.5 µL</td>
+            <td class="notTD">1141.25 µL</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD" style="width:33%">Total volume</td>
+            <td class="notTD">7700 µL</td>
+            <td class="notTD">1655.5 µL</td>
+            <td class="notTD">4427.5 µL</td>
+            <td class="notTD">1597.75 µL</td>
+          </tr>
+        </table>
+        <br>
+        <p class="p2N" style="text-indent:50px;">
+. &nbsp;&nbsp;Place the tube at -20°C overnight to precipitate the DNA from the sample. The experiment was continued on 08.02.19
+        </p>
+        <br><br>
+        <p class="date">
+.2.19
+        </p>
+        <br>
+        <p class="p3N">
+          DNA precipitation
+        </p>
+        <p class="p2N">
+. &nbsp;&nbsp;&nbsp;Centrifuge the sample at 4°C for 30 minutes at 16,000 × g to pellet the cDNA.
+. &nbsp;&nbsp;&nbsp;Carefully remove the supernatant without disturbing the cDNA pellet.
+. &nbsp;&nbsp;&nbsp;Add 150 μL of 70% ethanol.
+. &nbsp;&nbsp;&nbsp;Centrifuge the sample at 4°C for 15 minutes at 16,000 × g. Carefully remove the supernatant.
+. &nbsp;&nbsp;&nbsp;Dry the cDNA pellet at room temperature
+. &nbsp;&nbsp;&nbsp;Resuspend the cDNA pellet in 300 μL of TEN buffer by pipetting up and down
+        </p>
+        <br>
+        <p class="p3N">
+          Purification of PCR product &nbsp;-&nbsp; illustra MicroSpin G-25 Columns kit from GE Healthcare - PCR 2
+        </p>
+        <p class="p2N">
+          Because the purification by gel extraction was not conclusive, we tested a second method of purification using the illustra MicroSpin G-25 Columns kit from GE Healthcare. The experiment was performed according to the manufacturer’s recommendation.
+        </p>
+        <br><br>
+        <p class="p3N">
+          Gel electrophoresis 5 &nbsp;-&nbsp; Purified products from gel extraction vs. columns
+        </p>
+        <p class="p2N">
+          Because the purification by gel extraction was not conclusive, we tested a second method of purification using the illustra MicroSpin G-25 Columns kit from GE Healthcare. The experiment was performed according to the manufacturer’s recommendation.
+        </p>
+        <div>
+          <img src="https://2019.igem.org/wiki/images/e/e1/T--Pasteur_Paris--NotebookImg5.png" style="width:70%;margin-left:15%">
+        </div>
+        <br><br>
+        <p class="p3N">
+          Interpretation
+        </p>
+        <p class="p2N">
+          We only observed the bands from the samples purified with the columns. Moreover, they seemed well purified because there was almost no smear.
+        </p>
+        <br>
+        <p class="p3N">
+          PCR 3 &nbsp;-&nbsp; Testing different conditions (with/without DMSO + new program)
+        </p>
+        <p class="p2N">
+          To try to enhance our PCR protocol, we performed a new PCR using two conditions, one with DMSO and the other without . The PCR was run overnight and hold at 4°C.
+        </p>
+        <br>
+        <table class="notTABLE" style="border-color:white">
+          <tr class="notTR">
+            <td></td>
+            <td colspan="2" class="notTD">Volume needed (in &mu;L)</td>
+            <td></td>
+          </tr>
+          <tr class="NotTR">
+            <td class="notTD">Components</td>
+            <td class="notTD">Mix 1 (With DMSO)</td>
+            <td class="notTD">Mix 2 (Without DMSO)</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Phusion HF buffer 5X</td>
+            <td class="notTD">50</td>
+            <td class="notTD">50</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">25 mM dNTPs</td>
+            <td class="notTD">5</td>
+            <td class="notTD">5</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Forward primer</td>
+            <td class="notTD">10</td>
+            <td class="notTD">10</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Reverse primer</td>
+            <td class="notTD">10</td>
+            <td class="notTD">10</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">DMSO</td>
+            <td class="notTD">7.5</td>
+            <td class="notTD">0</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Template DNA</td>
+            <td class="notTD">22.5</td>
+            <td class="notTD">22.5</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Phusion DNA polymerase</td>
+            <td class="notTD">2.5</td>
+            <td class="notTD">2.5</td>
+          </tr>
+           <tr class="notTR">
+            <td class="notTD">Nuclease-free water</td>
+            <td class="notTD">142.5</td>
+            <td class="notTD">150</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Total volume</td>
+            <td class="notTD">250</td>
+            <td class="notTD">250</td>
+          </tr>
+        </table>
          </div>
        </div>
@@ Line 1,149: / Line 1,935: @@
            </div>
          </div>
-         <div id="AU1" class="ent" style="overflow-x:hidden;overflow:scroll;width:85%;margin-left:7.5%;opacity:0;transition:0.3s;">
+         <div id="AU1" class="ent"  >
-           <p style="color:black;font-family:roboto;font-size:20px;font-weight:100;text-align:justify;margin-right:5%;">
+           <p class="p1N">
-            Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
+          August Week 1 &nbsp;:&nbsp; 08/05 &nbsp;&nbsp;to&nbsp;&nbsp;08/11
-            purus augue, dictum non molestie eget, posuere a mauris. Donec
+        </p>
-            fringilla eros in ipsum molestie eleifend. Maecenas viverra ex sed
+        <p class="date">
-            dolor blandit, at finibus orci rhoncus. Aliquam non dui est. In
+.05.19
-             mattis tincidunt nisl, at egestas felis dignissim id. Nam
+        </p>
-             condimentum accumsan tellus quis ornare. Vestibulum quis augue
+        <br>
-             malesuada, tempor lorem ut, accumsan purus. Integer nibh nulla,
+        <p class="p3N">
-             malesuada ut sodales non, blandit non ligula. Donec venenatis
+          Purification of PCR products &nbsp;-&nbsp; illustra MicroSpin G-25 Columns kit from GE Healthcare &nbsp;-&nbsp; PCR 3
-             faucibus pulvinar. Sed tincidunt ultrices eros, nec cursus dui
+        </p>
-             molestie nec. Integer ac molestie lacus. Suspendisse lacinia mauris
+        <p class="p2N">
-             in dolor ullamcorper sodales. Lorem ipsum dolor sit amet,
+          Once again, we tried to purify our PCR products using different methods in order to compare them. This experiment was performed according to the manufacturer’s protocol.
-             consectetur adipiscing elit. Pellentesque ut elit vitae neque luctus
+        </p>
-             pellentesque porta sit amet orci.
+        <br>
-             Sed eget ante ipsum. Morbi pharetra lacinia purus, vitae efficitur
+        <p class="p3N">
-             eros convallis vitae. Aenean in consequat tortor, vitae dapibus
+          Purification of PCR products &nbsp;-&nbsp; PCR clean-up kit from Quiagen &nbsp;-&nbsp; PCR 3
-             nisi. Nunc non imperdiet tortor. Curabitur aliquet facilisis tempus.
+        </p>
-             Integer viverra orci sit amet erat fringilla, sed interdum diam
+        <p class="p2N">
-             euismod. Ut ac vulputate orci. Nunc iaculis tellus quis massa
+          This experiment was performed according to the manufacturer’s protocol.
-             convallis semper. Nunc nec consectetur arcu. In lectus lectus,
+        </p><br>
-             tempor id scelerisque vitae, egestas at purus. Curabitur vel erat
+        <p class="p3N">
-             quis dui pretium lobortis nec a tellus.
+          Gel electrophoresis 6 &nbsp;-&nbsp; Purified products from gel extraction (30.07.19) vs. GE Healthcare vs. Quiagen
-             Duis at efficitur sem. Suspendisse potenti. Donec pretium egestas
+        </p>
-             suscipit. Vivamus in vulputate urna. Etiam ullamcorper aliquam
+        <p class="p2N">
-             blandit. Phasellus accumsan volutpat tortor non dignissim. Nunc
+          To analyze the different purification methods of our PCR products from 02.08 and the effect of DMSO, we ran the samples on an agarose gel. Moreover, we compared the percentage of agarose in gel between 3 and 4% to see if the band resolution was better.
-             vestibulum tellus vel ex pulvinar convallis. Etiam ultricies
+        </p>
-             convallis malesuada. Praesent sed cursus lorem, vitae iaculis ipsum.
+        <div>
-            Vestibulum risus risus, vulputate non metus non, posuere finibus
+          <img src="https://2019.igem.org/wiki/images/7/78/T--Pasteur_Paris--NotebookImg6.png" style="width:70%;margin-left:15%">
-            magna. Nulla hendrerit lorem ac lacus ornare porta. Vivamus ligula
+        </div>
-            lorem, ullamcorper eu nisl eget, bibendum condimentum dui.
+        <br>
-            Aenean consectetur tincidunt ullamcorper. Vestibulum tempus, mi nec
+        <p class="p2N" style="border:1px solid black:margin-left:50px;">
-             gravida euismod, massa est consectetur ligula, vitae mattis nunc
+. &nbsp;&nbsp;&nbsp;PCR product from 02.08 with DMSO
-             mauris vel tortor. Vivamus ac mauris ac odio mollis volutpat vitae
+. &nbsp;&nbsp;&nbsp;PCR product from 02.08 without DMSO
-             ac felis. Proin eu ex et erat commodo vulputate. Morbi quis velit
+. &nbsp;&nbsp;&nbsp;PCR product with DMSO purified with Qiagen kit
-             eleifend libero finibus hendrerit. Mauris egestas ultrices odio in
+. &nbsp;&nbsp;&nbsp;PCR product whithout DMSO purified with Qiagen kit
-             rutrum. Pellentesque ut laoreet purus. In vitae orci ac tellus
+. &nbsp;&nbsp;&nbsp;PCR product with DMSO purified with GE Healthcare column
-             eleifend condimentum. Phasellus at purus quis orci tempor convallis
+. &nbsp;&nbsp;&nbsp;PCR product without DMSO purified with GE Healthcare column
-             vitae non nibh.
+. &nbsp;&nbsp;&nbsp;PCR product with DMSO purified by gel extraction
-             Cras hendrerit interdum lectus vel dictum. Donec nec lectus mattis,
+. &nbsp;&nbsp;&nbsp;PCR product without DMSO purified by gel exctraction
-             aliquam tortor vitae, convallis sem. Suspendisse vitae dui tortor.
+        </p>
-             Lorem ipsum dolor sit amet, consectetur adipiscing elit. Suspendisse
+        <br>
-             egestas, lacus a lacinia molestie, leo nibh malesuada velit, sed
+        <table class="notTABLE">
-             iaculis nulla libero nec dui. Maecenas fringilla, massa ut pretium
+          <tr class="NotTR">
-             hendrerit, lorem urna tristique ligula, sed pharetra eros diam vel
+             <td class="notTD">Wells</td>
-             odio. Aenean consectetur eu massa id volutpat. Nunc vel ex laoreet,
+            <td class="notTD">Purification method</td>
-             aliquam augue ac, ullamcorper nunc. Pellentesque ut tempor dolor, ac
+            <td class="notTD">Concentration (ng/&mu;L)</td>
-             eleifend ante.
+            <td class="notTD">260/280 ratio</td>
-             Pellentesque accumsan mauris in ante venenatis, nec elementum enim
+            <td class="notTD">260/230 ratio</td>
-             ornare. In varius rutrum odio imperdiet ultricies. Pellentesque nec
+          </tr>
-             feugiat tortor. Donec euismod leo orci, quis sodales felis placerat
+          <tr class="notTR">
-             ac. Donec a nibh dapibus, vulputate elit non, pretium nisl. Sed at
+            <td class="notTD">4</td>
-             est massa. Fusce at semper tortor. In cursus lorem lectus, nec
+            <td class="notTD">PCR product with DMSO</td>
-             suscipit nibh euismod eu. Morbi malesuada, lacus ut pellentesque
+            <td class="notTD">1157.6</td>
-             semper, diam tellus vulputate lectus, et varius ligula velit vitae
+             <td class="notTD">1.64</td>
-             elit. Praesent gravida, ex sit amet ornare bibendum, metus turpis
+             <td class="notTD">0.85</td>
-             maximus ipsum, eget interdum elit urna eu quam. Fusce tempor eu elit
+          </tr>
-             id efficitur. Curabitur vitae lorem vel arcu posuere blandit at et
+          <tr class="notTR">
-             dolor. Nunc posuere pharetra odio vitae iaculis. In blandit dolor
+             <td class="notTD">5</td>
-             quis magna cursus aliquet. Aliquam luctus hendrerit mauris ut
+            <td class="notTD">PCR product without DMSO</td>
-             tristique. Vivamus pharetra scelerisque nisl vel consequat.
+            <td class="notTD">1143</td>
-             Integer iaculis enim et dui cursus lacinia. Fusce nec ultricies
+            <td class="notTD">1.65</td>
-             quam, quis molestie augue. Cras mauris quam, eleifend quis sagittis
+             <td class="notTD">1.18</td>
-             eu, venenatis non mauris. Nulla sollicitudin ut nisl at ultrices.
+          </tr>
-             Vivamus fermentum placerat purus et gravida. Integer vulputate quam
+          <tr class="notTR">
-             nec nunc porttitor, vel gravida est aliquam. Aenean elementum eget
+             <td class="notTD">6</td>
-             velit ut tincidunt. Fusce sollicitudin, magna vitae sodales
+            <td class="notTD">PCR clean up kit from QIAGEN with DMSO</td>
-             tincidunt, diam turpis fermentum urna, eget maximus est libero
+            <td class="notTD">7.9</td>
-             lacinia nibh. Donec sagittis elementum molestie. Donec suscipit
+            <td class="notTD">2.26</td>
-             maximus laoreet. Proin sed convallis enim. Nullam dignissim pharetra
+             <td class="notTD">-0.32</td>
-             dapibus. Aenean vel varius ipsum.
+          </tr>
-            Ut eget rutrum nunc, sit amet tempor arcu. Etiam a tempus magna.
+          <tr class="notTR">
-            Vivamus leo nunc, consectetur non sagittis euismod, eleifend in
+             <td class="notTD">7</td>
-            ante. Etiam imperdiet at dui vitae lobortis. Donec at mattis ipsum.
+            <td class="notTD">PCR clean up kit from QIAGEN without DMSO</td>
-             Nam ut ante condimentum, sodales mauris vel, tempor massa. Curabitur
+            <td class="notTD">15</td>
-             feugiat libero consectetur velit viverra, nec fermentum leo
+            <td class="notTD">2.07</td>
-             ultrices. Nulla laoreet in orci consequat condimentum. Donec et
+             <td class="notTD">-0.62</td>
-             posuere mauris, sagittis eleifend augue.
+          </tr>
-             Nunc venenatis enim in auctor rutrum. Nullam molestie arcu quis
+          <tr class="notTR">
-             turpis mollis mattis. Phasellus elementum lectus vitae odio
+             <td class="notTD">8</td>
-             tincidunt, vitae congue nisl volutpat. Fusce sodales fringilla neque
+            <td class="notTD">GE Healthcare columns with DMSO</td>
-             sed posuere. Morbi risus sem, imperdiet iaculis neque ac, malesuada
+            <td class="notTD">34.5</td>
-             porta lectus. Quisque congue vel lorem vel commodo. In sit amet
+             <td class="notTD">0.96</td>
-            gravida purus. Aliquam erat volutpat.
+             <td class="notTD">0.34</td>
-             Praesent pellentesque pharetra sem sed condimentum. Quisque a
+          </tr>
-             bibendum turpis, et malesuada ipsum. Nunc viverra quam in magna
+          <tr class="notTR">
-             viverra facilisis. Ut pharetra eget urna sit amet luctus. Phasellus
+             <td class="notTD">9</td>
-             condimentum fermentum tortor, ut venenatis augue suscipit non.
+             <td class="notTD">GE Healthcare columns without DMSO</td>
-             Maecenas facilisis arcu non diam vehicula lobortis. Suspendisse ut
+            <td class="notTD">45.9</td>
-             neque libero. Quisque sagittis iaculis faucibus. Nam sagittis,
+             <td class="notTD">1.16</td>
-             mauris ut vestibulum tincidunt, dui ante sagittis ex, id finibus
+             <td class="notTD">0.08</td>
-             eros augue eu velit. Maecenas vulputate ante sed hendrerit
+          </tr>
-             vestibulum. Lorem ipsum dolor sit amet, consectetur adipiscing elit.
+          <tr class="notTR">
-             Nam enim purus, porta in leo id, accumsan gravida augue. Vestibulum
+             <td class="notTD">10</td>
-             quis sollicitudin lectus, a pretium erat. Donec hendrerit imperdiet
+            <td class="notTD">Gel extraction kit with DMSO</td>
-             turpis, vel convallis est pharetra quis. Curabitur molestie
+            <td class="notTD">6.1</td>
-             porttitor turpis, in fringilla arcu lacinia et. </p>
+             <td class="notTD">-6.66</td>
+             <td class="notTD">0.01</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">11</td>
+            <td class="notTD">Gel extraction kit without DMSO</td>
+            <td class="notTD">-0.5</td>
+             <td class="notTD">0.53</td>
+             <td class="notTD">0</td>
+          </tr>
+        </table>
+        <br>
+        <p class="p3N">
+          Interpretations
+        </p>
+        <p class="p2N">
+          The gel containing 4% of agarose gave a better resolution of the bands. Moreover, we did not observe any difference between the amplified products with and without DMSO. Concerning the purification methods, there was still no bands after the purification by gel extraction. Concerning the purification by PCR clean-up, we did not see anything on the gel at 4% agarose and there was only one band in the well 9 on the gel with 3% of agarose. On the contrary, we observed a band on both gels  for the two products purified by the columns.
+        </p>
+        <p class="p2N">
+          We analyzed then the same products as above but after digestion. The digestion was performed according to the manufacturer’s protocol.  This time we only did a 4% of agarose gel because we noticed with the previous experiment that we had a better resolution with this concentration.
+        </p>
+        <div>
+          <img src="https://2019.igem.org/wiki/images/2/25/T--Pasteur_Paris--NotebookImg7.png" style="width:70%;margin-left:10%">
+        </div>
+        <br>
+        <p class="p3N">
+          Interpretation
+        </p>
+        <p class="p2N">
+          After digestion, there was no more bands even for the sample purified with the columns. Thus, either the lambda exonuclease had digested the two strands of DNA, or the concentration of DNA was too low to be seen on the gel. The last hypothesis was that the SYBR Green may not reveal single-stranded DNA.
+        </p>
+        <br>
+        <p class="p3N">
+          Conclusion
+        </p>
+        <p class="p2N">
+          Several parameters may change during the digestion step in order to determine if the enzyme digested the two strands of DNA.
+        </p>
+        <br>
+        <p class="p3N">
+          Phenol/chloroform extraction
+        </p>
+        <p class="p2N">
+          We performed a purification step on all samples to remove the digestion enzyme according to the protocol previously described. Samples were then analyzed using a Nanodrop 2000 spectrophotometer.
+        </p>
+        <br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+             <td class="notTD">Wells</td>
+             <td class="notTD">Purification method</td>
+             <td class="notTD">Concentration (ng/&mu;L)</td>
+             <td class="notTD">260/280 ratio</td>
+             <td class="notTD">260/230 ratio</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">5</td>
+             <td class="notTD">PCR product with DMSO</td>
+             <td class="notTD">1157.6</td>
+             <td class="notTD">1.64</td>
+             <td class="notTD">0.85</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">6</td>
+             <td class="notTD">PCR product without DMSO</td>
+             <td class="notTD">1143</td>
+             <td class="notTD">1.65</td>
+             <td class="notTD">1.18</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">8</td>
+             <td class="notTD">PCR product clean up kit from QIAGEN with DMSO</td>
+             <td class="notTD">7.9</td>
+             <td class="notTD">2.26</td>
+             <td class="notTD">-0.32</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">9</td>
+             <td class="notTD">PCR product clean up kit from QIAGEN without DMSO</td>
+             <td class="notTD">15</td>
+             <td class="notTD">2.07</td>
+             <td class="notTD">-0.62</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">10</td>
+             <td class="notTD">GE Healthcare columns with DMSO</td>
+            <td class="notTD">34.5</td>
+             <td class="notTD">0.96</td>
+             <td class="notTD">0.34</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">11</td>
+            <td class="notTD">GE Healthcare columns without DMSO</td>
+            <td class="notTD">45.9</td>
+             <td class="notTD">1.16</td>
+             <td class="notTD">0.08</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">13</td>
+            <td class="notTD">Gel extraction kit with DMSO</td>
+            <td class="notTD">6.1</td>
+             <td class="notTD">-6.66</td>
+             <td class="notTD">0.01</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">14</td>
+             <td class="notTD">Gel extraction kit without DMSO</td>
+            <td class="notTD">-0.5</td>
+             <td class="notTD">0.53</td>
+             <td class="notTD">0</td>
+          </tr>
+        </table>
+        <br>
+        <p class="date">
+.08.19
+        </p>
+        <br>
+        <p class="p3N">
+          PCR 4 &nbsp;-&nbsp; 15 cycles vs 30 cycles
+        </p>
+        <p class="p2N">
+          We tried to amplify our library with only 15 cycles instead of 30 to compare if it had an impact in the shift of size of our PCR product (120 bp instead of 80 bp normally). The PCR was made with the same conditions as the PCR 4 without DMSO.
+        </p>
+        <p class="p3N">
+          Gel electrophoresis 7 &nbsp;-&nbsp; 15 cycles vs 30 cycles
+        </p>
+        <p class="p2N">
+          The amplified products were run on a 4% agarose gel and compared with amplified products from PCR 3 and 4.
+        </p>
+        <br>
+        <div>
+          <img src="https://2019.igem.org/wiki/images/4/4b/T--Pasteur_Paris--NotebookImg8.png" style="width:70%;margin-left:10%">
+        </div>
+        <p class="p3N">
+          Interpretations
+        </p>
+        <p class="p2N">
+          We observed a slight decrease of the size for products amplified with only 15 cycles compared to those amplified with 30 cycles. However, this decrease in size was not significant enough. And the bands from the amplified samples with 30 cycles were brighter than those amplified with 15 cycles.
+        </p>
+        <br>
+        <p class="p3N">
+          Purification of PCR products &nbsp;-&nbsp; Dialysis tubing
+        </p>
+        <p class="p2N">
+          A new purification method of our PCR products was tested which is dialysis tubing. The DNA will migrate out of the gel and into the dialysis bag. Samples were checked using a Nanodrop 2000 spectrophotometer.
+        </p>
+        <br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+          	<td class="notTD">Sample</td>
+             <td class="notTD">Concentration (ng/&mu;L)</td>
+             <td class="notTD">260/280 ratio</td>
+             <td class="notTD">260/230 ratio</td>
+          </tr>
+           <tr class="notTR">
+          	<td class="notTD">Dialysis sample 1 pool</td>
+             <td class="notTD">1.2</td>
+             <td class="notTD">2.71</td>
+             <td class="notTD">-9.20</td>
+          </tr>
+           <tr class="notTR">
+          	<td class="notTD">Dialysis sample 2 pool</td>
+             <td class="notTD">2.8</td>
+             <td class="notTD">2.22</td>
+             <td class="notTD">0.6</td>
+          </tr>
+        </table>
+        <br>
+        <p class="p3N">
+          Interpretation
+        </p>
+        <p class="p2N">
+          Even if the ratio 260/280 was good the concentration of DNA was too low. Thus, this technique cannot be used due to a yield too low.
+        </p>
+        <br>
+        <p class="p3N">
+          Kit montage Gel Extraction
+        </p>
+        <p class="p2N">
+          We used the Kit montage Gel extraction from Millipore to try to extract our DNA from the gel and purify it. This kit was used according to the manufacturer’s protocol. Samples were checked using a Nanodrop 2000 spectrophotometer.
+        </p>
+        <br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+             <td class="notTD">Sample</td>
+            <td class="notTD">Concentration (ng/&mu;L)</td>
+            <td class="notTD">260/280 ratio</td>
+            <td class="notTD">260/230 ratio</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Sample 1</td>
+            <td class="notTD">8.3</td>
+             <td class="notTD">1.95</td>
+             <td class="notTD">0.15</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 2</td>
+            <td class="notTD">6.4</td>
+             <td class="notTD">2.13</td>
+             <td class="notTD">0.16</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 3</td>
+             <td class="notTD">5</td>
+             <td class="notTD">2.24</td>
+             <td class="notTD">0.12</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 4</td>
+            <td class="notTD">6.9</td>
+             <td class="notTD">1.37</td>
+             <td class="notTD">0.14</td>
+          </tr>
+        </table>
+        <br>
+        <p class="p3N">
+          Interpretation
+        </p>
+        <p class="p2N">
+          The concentration of DNA was too low. Hence, another method of DNA purification has to be found.
+        </p>
          </div>
        </div>
@@ Line 1,255: / Line 2,260: @@
            </div>
          </div>
-         <div id="AU2" class="ent" style="overflow-x:hidden;overflow:scroll;width:85%;margin-left:7.5%;opacity:0;transition:0.3s;">
+         <div id="AU2" class="ent"  >
-           <p style="color:black;font-family:roboto;font-size:20px;font-weight:100;text-align:justify;margin-right:5%;">
+                   <p class="p1N"> August Week 2 : &nbsp;07/12 &nbsp;to &nbsp;07/18 </p>
-            Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
+        <p class="date"> 07.12.19 </p>
-            purus augue, dictum non molestie eget, posuere a mauris. Donec
+        <p class="p3N"> Digestion &nbsp;-&nbsp; Testing different conditions on
-             fringilla eros in ipsum molestie eleifend. Maecenas viverra ex sed
+          amplified library </p>
-             dolor blandit, at finibus orci rhoncus. Aliquam non dui est. In
+        <p class="p2N"> A new digestion was carried out in order to optimize the
-             mattis tincidunt nisl, at egestas felis dignissim id. Nam
+          yield. Two parameters were studied, the incubation time and the
-            condimentum accumsan tellus quis ornare. Vestibulum quis augue
+          quantity of enzyme used. Here is the list of all conditions tested: </p>
-            malesuada, tempor lorem ut, accumsan purus. Integer nibh nulla,
+        <li class="notLI">1A : 5 min incubation, 5 U</li>
-             malesuada ut sodales non, blandit non ligula. Donec venenatis
+        <li class="notLI">1B : 5 min incubation, 2.5 U</li>
-             faucibus pulvinar. Sed tincidunt ultrices eros, nec cursus dui
+        <li class="notLI">1C : 5 min incubation, 1 U</li>
-            molestie nec. Integer ac molestie lacus. Suspendisse lacinia mauris
+        <li class="notLI">2A : 10 min incubation, 5 U</li>
-             in dolor ullamcorper sodales. Lorem ipsum dolor sit amet,
+        <li class="notLI">2B : 10 min incubation, 2.5 U</li>
-             consectetur adipiscing elit. Pellentesque ut elit vitae neque luctus
+        <li class="notLI">2C : 10 min incubation, 1 U</li>
-            pellentesque porta sit amet orci.
+        <li class="notLI">3A : 15 min incubation, 5 U</li>
-             Sed eget ante ipsum. Morbi pharetra lacinia purus, vitae efficitur
+        <li class="notLI">3B : 15 min incubation, 2.5 U</li>
-            eros convallis vitae. Aenean in consequat tortor, vitae dapibus
+        <li class="notLI">3C : 15 min incubation, 1 U</li>
-            nisi. Nunc non imperdiet tortor. Curabitur aliquet facilisis tempus.
+        <li class="notLI">4A : 20 min incubation, 5 U</li>
-             Integer viverra orci sit amet erat fringilla, sed interdum diam
+        <li class="notLI">4B : 20 min incubation, 2.5 U</li>
-             euismod. Ut ac vulputate orci. Nunc iaculis tellus quis massa
+        <li class="notLI">4C : 20 min incubation, 1 U</li>
-            convallis semper. Nunc nec consectetur arcu. In lectus lectus,
+        <p></p>
-             tempor id scelerisque vitae, egestas at purus. Curabitur vel erat
+        <p class="p3N"> Gel electrophoresis 8 &nbsp;-&nbsp; Digestion conditions
-             quis dui pretium lobortis nec a tellus.
+          comparison </p>
-            Duis at efficitur sem. Suspendisse potenti. Donec pretium egestas
+        <br>
-             suscipit. Vivamus in vulputate urna. Etiam ullamcorper aliquam
+        <div> <img src="https://2019.igem.org/wiki/images/9/94/T--Pasteur_Paris--NotebookImg9.png"
-             blandit. Phasellus accumsan volutpat tortor non dignissim. Nunc
-             vestibulum tellus vel ex pulvinar convallis. Etiam ultricies
+            style="width:70%;margin-left:10%"> </div>
-             convallis malesuada. Praesent sed cursus lorem, vitae iaculis ipsum.
+        <br>
-             Vestibulum risus risus, vulputate non metus non, posuere finibus
+        <p class="p2N"> Samples were then checked using a Nanodrop 2000
-             magna. Nulla hendrerit lorem ac lacus ornare porta. Vivamus ligula
+          spectrophotometer. </p>
-            lorem, ullamcorper eu nisl eget, bibendum condimentum dui.
+        <br>
-            Aenean consectetur tincidunt ullamcorper. Vestibulum tempus, mi nec
+        <table class="notTABLE">
-             gravida euismod, massa est consectetur ligula, vitae mattis nunc
+          <tbody>
-             mauris vel tortor. Vivamus ac mauris ac odio mollis volutpat vitae
+             <tr class="NotTR">
-            ac felis. Proin eu ex et erat commodo vulputate. Morbi quis velit
+              <td class="notTD">Sample</td>
-             eleifend libero finibus hendrerit. Mauris egestas ultrices odio in
+              <td class="notTD">Concentration (ng/µl)</td>
-             rutrum. Pellentesque ut laoreet purus. In vitae orci ac tellus
+              <td class="notTD">260/280 ratio</td>
-            eleifend condimentum. Phasellus at purus quis orci tempor convallis
+              <td class="notTD">260/230 ratio</td>
-             vitae non nibh.
+             </tr>
-             Cras hendrerit interdum lectus vel dictum. Donec nec lectus mattis,
+             <tr class="notTR">
-            aliquam tortor vitae, convallis sem. Suspendisse vitae dui tortor.
+              <td class="notTD">1A after digestion</td>
-            Lorem ipsum dolor sit amet, consectetur adipiscing elit. Suspendisse
+              <td class="notTD">121.4</td>
-            egestas, lacus a lacinia molestie, leo nibh malesuada velit, sed
+              <td class="notTD">1.52</td>
-            iaculis nulla libero nec dui. Maecenas fringilla, massa ut pretium
+              <td class="notTD">0.29</td>
-            hendrerit, lorem urna tristique ligula, sed pharetra eros diam vel
+             </tr>
-            odio. Aenean consectetur eu massa id volutpat. Nunc vel ex laoreet,
+             <tr class="notTR">
-            aliquam augue ac, ullamcorper nunc. Pellentesque ut tempor dolor, ac
+              <td class="notTD">1B after digestion</td>
-            eleifend ante.
+              <td class="notTD">117.2</td>
-            Pellentesque accumsan mauris in ante venenatis, nec elementum enim
+              <td class="notTD">1.52</td>
-            ornare. In varius rutrum odio imperdiet ultricies. Pellentesque nec
+              <td class="notTD">0.29</td>
-            feugiat tortor. Donec euismod leo orci, quis sodales felis placerat
+             </tr>
-            ac. Donec a nibh dapibus, vulputate elit non, pretium nisl. Sed at
+             <tr class="notTR">
-            est massa. Fusce at semper tortor. In cursus lorem lectus, nec
+              <td class="notTD">1C after digestion</td>
-            suscipit nibh euismod eu. Morbi malesuada, lacus ut pellentesque
+              <td class="notTD">118</td>
-            semper, diam tellus vulputate lectus, et varius ligula velit vitae
+              <td class="notTD">1.55</td>
-            elit. Praesent gravida, ex sit amet ornare bibendum, metus turpis
+              <td class="notTD">0.29</td>
-            maximus ipsum, eget interdum elit urna eu quam. Fusce tempor eu elit
+             </tr>
-             id efficitur. Curabitur vitae lorem vel arcu posuere blandit at et
+             <tr class="notTR">
-             dolor. Nunc posuere pharetra odio vitae iaculis. In blandit dolor
+              <td class="notTD">2A after digestion</td>
-             quis magna cursus aliquet. Aliquam luctus hendrerit mauris ut
+              <td class="notTD">69.3</td>
-             tristique. Vivamus pharetra scelerisque nisl vel consequat.
+              <td class="notTD">1.64</td>
-             Integer iaculis enim et dui cursus lacinia. Fusce nec ultricies
+              <td class="notTD">0.3</td>
-             quam, quis molestie augue. Cras mauris quam, eleifend quis sagittis
+             </tr>
-             eu, venenatis non mauris. Nulla sollicitudin ut nisl at ultrices.
+             <tr class="notTR">
-             Vivamus fermentum placerat purus et gravida. Integer vulputate quam
+              <td class="notTD">2B after digestion</td>
-             nec nunc porttitor, vel gravida est aliquam. Aenean elementum eget
+              <td class="notTD">116.5</td>
-             velit ut tincidunt. Fusce sollicitudin, magna vitae sodales
+              <td class="notTD">1.49</td>
-             tincidunt, diam turpis fermentum urna, eget maximus est libero
+              <td class="notTD">0.3</td>
-             lacinia nibh. Donec sagittis elementum molestie. Donec suscipit
+             </tr>
-             maximus laoreet. Proin sed convallis enim. Nullam dignissim pharetra
+             <tr class="notTR">
-             dapibus. Aenean vel varius ipsum.
+              <td class="notTD">2C after digestion</td>
-             Ut eget rutrum nunc, sit amet tempor arcu. Etiam a tempus magna.
+              <td class="notTD">117.3</td>
-             Vivamus leo nunc, consectetur non sagittis euismod, eleifend in
+              <td class="notTD">1.51</td>
-             ante. Etiam imperdiet at dui vitae lobortis. Donec at mattis ipsum.
+              <td class="notTD">0.28</td>
-             Nam ut ante condimentum, sodales mauris vel, tempor massa. Curabitur
+             </tr>
-             feugiat libero consectetur velit viverra, nec fermentum leo
+             <tr class="notTR">
-             ultrices. Nulla laoreet in orci consequat condimentum. Donec et
+              <td class="notTD">3A after digestion</td>
-             posuere mauris, sagittis eleifend augue.
+              <td class="notTD">119</td>
-             Nunc venenatis enim in auctor rutrum. Nullam molestie arcu quis
+              <td class="notTD">1.55</td>
-             turpis mollis mattis. Phasellus elementum lectus vitae odio
+              <td class="notTD">0.28</td>
-             tincidunt, vitae congue nisl volutpat. Fusce sodales fringilla neque
+             </tr>
-             sed posuere. Morbi risus sem, imperdiet iaculis neque ac, malesuada
+             <tr class="notTR">
-             porta lectus. Quisque congue vel lorem vel commodo. In sit amet
+              <td class="notTD">3B after digestion</td>
-             gravida purus. Aliquam erat volutpat.
+              <td class="notTD">110.6</td>
-             Praesent pellentesque pharetra sem sed condimentum. Quisque a
+              <td class="notTD">1.54</td>
-             bibendum turpis, et malesuada ipsum. Nunc viverra quam in magna
+              <td class="notTD">0.28</td>
-             viverra facilisis. Ut pharetra eget urna sit amet luctus. Phasellus
+             </tr>
-             condimentum fermentum tortor, ut venenatis augue suscipit non.
+             <tr class="notTR">
-             Maecenas facilisis arcu non diam vehicula lobortis. Suspendisse ut
+              <td class="notTD">3C after digestion</td>
-             neque libero. Quisque sagittis iaculis faucibus. Nam sagittis,
+              <td class="notTD">117.4</td>
-             mauris ut vestibulum tincidunt, dui ante sagittis ex, id finibus
+              <td class="notTD">1.57</td>
-             eros augue eu velit. Maecenas vulputate ante sed hendrerit
+              <td class="notTD">0.24</td>
-             vestibulum. Lorem ipsum dolor sit amet, consectetur adipiscing elit.
+             </tr>
-             Nam enim purus, porta in leo id, accumsan gravida augue. Vestibulum
+             <tr class="notTR">
-             quis sollicitudin lectus, a pretium erat. Donec hendrerit imperdiet
+              <td class="notTD">4A after digestion</td>
-             turpis, vel convallis est pharetra quis. Curabitur molestie
+              <td class="notTD">120.5</td>
-             porttitor turpis, in fringilla arcu lacinia et. </p>
+              <td class="notTD">1.52</td>
+              <td class="notTD">0.26</td>
+             </tr>
+             <tr class="notTR">
+              <td class="notTD">4B after digestion</td>
+              <td class="notTD">118.3</td>
+              <td class="notTD">1.56</td>
+              <td class="notTD">0.24</td>
+             </tr>
+             <tr class="notTR">
+              <td class="notTD">4C after digestion</td>
+              <td class="notTD">130.1</td>
+              <td class="notTD">1.52</td>
+              <td class="notTD">0.26</td>
+             </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="p3N">
+          Interpretations
+        </p>
+        <p class="p2N">
+          There was no bands on the gel. However, the nanodrop detected DNA with a pretty good 260/280 ratio. But there was no difference between the different conditions. So, either the single-strand DNA i s not revealed with SYBR Green or the digestion enzyme does not work properly.
+        </p>
+        <br>
+        <p class="date">
+.14.19
+        </p>
+        <p class="p3N">
+          Denaturation &nbsp;-&nbsp; of PCR 5 products
+        </p>
+        <p class="p2N">
+          In order to optimize the digestion step, we tried to denature our PCR product before being digested. Samples were heated at 95°C for 5 min and directly put in ice for 15 min.
+        </p>
+        <br>
+        <p  class="p3N">
+          Digestion &nbsp;-&nbsp; Testing different conditions on PCR 5 products
+        </p>
+        <p class="p2N">
+          Different conditions of digestion were tested in order to optimize this step (buffers, DNA quantity to digest, enzyme quantity, with/without denaturation before).
+          Conditions tested:
+          <li class="notLI">Cutsmart, 15 µg DNA, 1 U (37°C, 15 min)</li>
+          <li class="notLI">Cutsmart, 10 µg DNA, 1 U (37°C, 15 min)</li>
+          <li class="notLI">Cutsmart, 15 µg DNA, 0,5 U (37°C, 15 min)</li>
+          <li class="notLI">Cutsmart, 10 µg DNA, 0,5 U (37°C, 15 min)</li>
+          <li class="notLI">Lambda buffer, 15 µg DNA, 1 U (37°C, 15 min)</li>
+          <li class="notLI">Lambda buffer, 10 µg DNA, 1 U (37°C, 15 min)</li>
+          <li class="notLI">Lambda buffer, 15 µg DNA, 0,5 U (37°C, 15 min)</li>
+          <li class="notLI">Lambda buffer, 10 µg DNA, 0,5 U (37°C, 15 min)</li>
+          <li class="notLI">Lambda buffer, 15 µg DNA, 1 U (37°C, 15 min) + denaturation before</li>
+          <li class="notLI">Lambda buffer, 10 µg DNA, 1 U (37°C, 15 min) + denaturation before</li>
+          <li class="notLI">Lambda buffer, 15 µg DNA, 0,5 U (37°C, 15 min) + denaturation before</li>
+          <li class="notLI">Lambda buffer, 10 µg DNA, 0,5 U (37°C, 15 min) + denaturation before</li>
+          <li class="notLI">50 bp ladder</li>
+          <li class="notLI">PCR product before digestion</li>
+          <li class="notLI">PCR product before digestion followed by heat shock</li>
+        </p>
+        <br>
+        <p class="p3N">
+          Gel electrophoresis 9 &nbsp;-&nbsp; Digestion conditions comparison
+        </p>
+        <p class="p2N">
+          A 4% agarose gel was made to compare all the different conditions of digestion.
+        </p>
+        <div>
+          <img src="https://2019.igem.org/wiki/images/3/3b/T--Pasteur_Paris--NotebookImg10.png" style="width:70%;margin-left:10%">
+        </div>
+        <br>
+        <p class="p3N">
+          Interpretations
+        </p>
+        <p class="p2N">
+          We are looking for a band with a shift in size compared to the PCR product before digestion. Once again, bands were similar to the PCR product, it means that conditions were too restrictive and that the digestion did not worked.
+        </p>
+        <br>
+        <p class="date">
+.15.19
+        </p>
+        <p class="p3N">
+          PCR 6 &nbsp;-&nbsp; Generation of new materials
+        </p>
+        <p class="p2N">
+          To continue our experiments, a new part of the library was amplified in order to work with. Samples were also analyzed using a Nanodrop 2000 spectrophotometer.
+        </p>
+        <br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+             <td class="notTD">Sample</td>
+             <td class="notTD">Concentration (ng/&mu;L)</td>
+             <td class="notTD">260/280 ratio</td>
+             <td class="notTD">260/230 ratio</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 1</td>
+            <td class="notTD">1231.1</td>
+             <td class="notTD">1.65</td>
+             <td class="notTD">1.15</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 2</td>
+            <td class="notTD">1217.1</td>
+             <td class="notTD">1.5</td>
+             <td class="notTD">1.15</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 3</td>
+             <td class="notTD">1219.1</td>
+             <td class="notTD">1.66</td>
+             <td class="notTD">1.16</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 4</td>
+             <td class="notTD">1215.3</td>
+             <td class="notTD">1.64</td>
+             <td class="notTD">1.15</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 5</td>
+             <td class="notTD">1216.2</td>
+             <td class="notTD">1.65</td>
+             <td class="notTD">1.15</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 6</td>
+             <td class="notTD">1213.2</td>
+             <td class="notTD">1.66</td>
+             <td class="notTD">1.14</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 7</td>
+             <td class="notTD">1201.4</td>
+             <td class="notTD">1.66</td>
+             <td class="notTD">1.14</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 8</td>
+            <td class="notTD">1211.5</td>
+             <td class="notTD">1.65</td>
+             <td class="notTD">1.15</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 9</td>
+             <td class="notTD">1221.2</td>
+             <td class="notTD">1.65</td>
+             <td class="notTD">1.14</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Sample 10</td>
+            <td class="notTD">1185.2</td>
+             <td class="notTD">1.65</td>
+             <td class="notTD">1.14</td>
+          </tr>
+        </table>
          </div>
        </div>
@@ Line 1,361: / Line 2,518: @@
            </div>
          </div>
-         <div id="AU3" class="ent" style="overflow-x:hidden;overflow:scroll;width:85%;margin-left:7.5%;opacity:0;transition:0.3s;">
+         <div id="AU3" class="ent"  >
-           <p style="color:black;font-family:roboto;font-size:20px;font-weight:100;text-align:justify;margin-right:5%;">
+          <p class="p1N"> August Week 3 &nbsp;: &nbsp;08/19 &nbsp;to
-            Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
+          &nbsp;08/21 <br>
-            purus augue, dictum non molestie eget, posuere a mauris. Donec
+          <br>
-             fringilla eros in ipsum molestie eleifend. Maecenas viverra ex sed
+        </p>
-             dolor blandit, at finibus orci rhoncus. Aliquam non dui est. In
+        <p class="date">08.19.19</p>
-             mattis tincidunt nisl, at egestas felis dignissim id. Nam
+        <p class="p3N">PCR 7&nbsp;- Generation of new materials (10 ng, 5 ng, 1
-            condimentum accumsan tellus quis ornare. Vestibulum quis augue
+          ng)</p>
-            malesuada, tempor lorem ut, accumsan purus. Integer nibh nulla,
+        <p class="p2N">A new PCR was performed to produce starting material. For
-            malesuada ut sodales non, blandit non ligula. Donec venenatis
+          this, different amounts of DNA to be amplified have been tested (10
-            faucibus pulvinar. Sed tincidunt ultrices eros, nec cursus dui
+          ng, 5 ng, 1 ng) in order to reduce nonspecific amplifications.</p>
-             molestie nec. Integer ac molestie lacus. Suspendisse lacinia mauris
+        <br>
-             in dolor ullamcorper sodales. Lorem ipsum dolor sit amet,
+        <table style="border-color: transparent" class="notTABLE">
-            consectetur adipiscing elit. Pellentesque ut elit vitae neque luctus
+          <tbody>
-            pellentesque porta sit amet orci.
+             <tr class="notTR">
-            Sed eget ante ipsum. Morbi pharetra lacinia purus, vitae efficitur
+              <td><br>
-            eros convallis vitae. Aenean in consequat tortor, vitae dapibus
+              </td>
-             nisi. Nunc non imperdiet tortor. Curabitur aliquet facilisis tempus.
+              <td colspan="3" class="notTD"> Volume needed (in μL)</td>
-             Integer viverra orci sit amet erat fringilla, sed interdum diam
+             </tr>
-            euismod. Ut ac vulputate orci. Nunc iaculis tellus quis massa
+             <tr class="NotTR">
-            convallis semper. Nunc nec consectetur arcu. In lectus lectus,
+              <td class="notTD">Components</td>
-            tempor id scelerisque vitae, egestas at purus. Curabitur vel erat
+              <td class="notTD">Mix 1(10ng)</td>
-            quis dui pretium lobortis nec a tellus.
+              <td class="notTD">Mix 2(5ng)</td>
-             Duis at efficitur sem. Suspendisse potenti. Donec pretium egestas
+              <td class="not TD">Mix 3(1ng)</td>
-             suscipit. Vivamus in vulputate urna. Etiam ullamcorper aliquam
+             </tr>
-            blandit. Phasellus accumsan volutpat tortor non dignissim. Nunc
+             <tr class="notTR">
-            vestibulum tellus vel ex pulvinar convallis. Etiam ultricies
+              <td class="notTD">Phusion Buffer 10X</td>
-            convallis malesuada. Praesent sed cursus lorem, vitae iaculis ipsum.
+              <td class="notTD">100</td>
-            Vestibulum risus risus, vulputate non metus non, posuere finibus
+              <td class="notTD">100</td>
-             magna. Nulla hendrerit lorem ac lacus ornare porta. Vivamus ligula
+              <td class="notTD">100</td>
-             lorem, ullamcorper eu nisl eget, bibendum condimentum dui.
+             </tr>
-            Aenean consectetur tincidunt ullamcorper. Vestibulum tempus, mi nec
+             <tr class="notTR">
-            gravida euismod, massa est consectetur ligula, vitae mattis nunc
+              <td class="notTD">dNTPs 25 mM</td>
-            mauris vel tortor. Vivamus ac mauris ac odio mollis volutpat vitae
+              <td class="notTD">10</td>
-            ac felis. Proin eu ex et erat commodo vulputate. Morbi quis velit
+              <td class="notTD">10</td>
-             eleifend libero finibus hendrerit. Mauris egestas ultrices odio in
+              <td class="notTD">10</td>
-             rutrum. Pellentesque ut laoreet purus. In vitae orci ac tellus
+             </tr>
-            eleifend condimentum. Phasellus at purus quis orci tempor convallis
+             <tr class="notTR">
-            vitae non nibh.
+              <td class="notTD">Forward primer (1/10)</td>
-            Cras hendrerit interdum lectus vel dictum. Donec nec lectus mattis,
+              <td class="notTD">20</td>
-            aliquam tortor vitae, convallis sem. Suspendisse vitae dui tortor.
+              <td class="notTD">20</td>
-             Lorem ipsum dolor sit amet, consectetur adipiscing elit. Suspendisse
+              <td class="notTD">20</td>
-             egestas, lacus a lacinia molestie, leo nibh malesuada velit, sed
+             </tr>
-            iaculis nulla libero nec dui. Maecenas fringilla, massa ut pretium
+             <tr class="notTR">
-            hendrerit, lorem urna tristique ligula, sed pharetra eros diam vel
+              <td class="notTD"> Reverse primer (1/10)</td>
-            odio. Aenean consectetur eu massa id volutpat. Nunc vel ex laoreet,
+              <td class="notTD">20</td>
-             aliquam augue ac, ullamcorper nunc. Pellentesque ut tempor dolor, ac
+              <td class="notTD">20</td>
-             eleifend ante.
+              <td class="notTD">20</td>
-            Pellentesque accumsan mauris in ante venenatis, nec elementum enim
+             </tr>
-             ornare. In varius rutrum odio imperdiet ultricies. Pellentesque nec
+             <tr class="notTR">
-             feugiat tortor. Donec euismod leo orci, quis sodales felis placerat
+              <td class="notTD"> Template DNA (1/1000)</td>
-             ac. Donec a nibh dapibus, vulputate elit non, pretium nisl. Sed at
+              <td class="notTD">45</td>
-            est massa. Fusce at semper tortor. In cursus lorem lectus, nec
+              <td class="notTD">22.5</td>
-            suscipit nibh euismod eu. Morbi malesuada, lacus ut pellentesque
+              <td class="notTD">5</td>
-            semper, diam tellus vulputate lectus, et varius ligula velit vitae
+             </tr>
-             elit. Praesent gravida, ex sit amet ornare bibendum, metus turpis
+             <tr class="notTR">
-            maximus ipsum, eget interdum elit urna eu quam. Fusce tempor eu elit
+              <td class="notTD">Phusion DNA polymerase</td>
-            id efficitur. Curabitur vitae lorem vel arcu posuere blandit at et
+              <td class="notTD">5</td>
-            dolor. Nunc posuere pharetra odio vitae iaculis. In blandit dolor
+              <td class="notTD">5</td>
-             quis magna cursus aliquet. Aliquam luctus hendrerit mauris ut
+              <td class="notTD">5</td>
-             tristique. Vivamus pharetra scelerisque nisl vel consequat.
+             </tr>
-            Integer iaculis enim et dui cursus lacinia. Fusce nec ultricies
+             <tr class="notTR">
-            quam, quis molestie augue. Cras mauris quam, eleifend quis sagittis
+              <td class="notTD">Nuclease-free water</td>
-             eu, venenatis non mauris. Nulla sollicitudin ut nisl at ultrices.
+              <td class="notTD">300</td>
-             Vivamus fermentum placerat purus et gravida. Integer vulputate quam
+              <td class="notTD">322.5</td>
-            nec nunc porttitor, vel gravida est aliquam. Aenean elementum eget
+              <td class="notTD">340</td>
-            velit ut tincidunt. Fusce sollicitudin, magna vitae sodales
+             </tr>
-             tincidunt, diam turpis fermentum urna, eget maximus est libero
+             <tr class="notTR">
-             lacinia nibh. Donec sagittis elementum molestie. Donec suscipit
+              <td class="notTD">Total volume</td>
-            maximus laoreet. Proin sed convallis enim. Nullam dignissim pharetra
+              <td class="notTD">500</td>
-            dapibus. Aenean vel varius ipsum.
+              <td class="notTD">500</td>
-            Ut eget rutrum nunc, sit amet tempor arcu. Etiam a tempus magna.
+              <td class="notTD">500</td>
-             Vivamus leo nunc, consectetur non sagittis euismod, eleifend in
+             </tr>
-            ante. Etiam imperdiet at dui vitae lobortis. Donec at mattis ipsum.
+          </tbody>
-            Nam ut ante condimentum, sodales mauris vel, tempor massa. Curabitur
+        </table>
-            feugiat libero consectetur velit viverra, nec fermentum leo
+        <p class="p2N">Samples were also analyzed using a Nanodrop 2000
-            ultrices. Nulla laoreet in orci consequat condimentum. Donec et
+          spectrophotometer.</p>
-            posuere mauris, sagittis eleifend augue.
+        <table class="notTABLE">
-            Nunc venenatis enim in auctor rutrum. Nullam molestie arcu quis
+          <tbody>
-            turpis mollis mattis. Phasellus elementum lectus vitae odio
+             <tr class="NotTR">
-            tincidunt, vitae congue nisl volutpat. Fusce sodales fringilla neque
+              <td class="notTD">Sample</td>
-            sed posuere. Morbi risus sem, imperdiet iaculis neque ac, malesuada
+              <td class="notTD">Concentration (ng/μl)</td>
-            porta lectus. Quisque congue vel lorem vel commodo. In sit amet
+              <td class="notTD">260/280 ratio</td>
-            gravida purus. Aliquam erat volutpat.
+              <td class="notTD">260/230 ratio</td>
-            Praesent pellentesque pharetra sem sed condimentum. Quisque a
+             </tr>
-             bibendum turpis, et malesuada ipsum. Nunc viverra quam in magna
+             <tr class="notTR">
-            viverra facilisis. Ut pharetra eget urna sit amet luctus. Phasellus
+              <td class="notTD">Sample pooled (10ng)</td>
-            condimentum fermentum tortor, ut venenatis augue suscipit non.
+              <td class="notTD">990.6</td>
-            Maecenas facilisis arcu non diam vehicula lobortis. Suspendisse ut
+              <td class="notTD">1.62</td>
-            neque libero. Quisque sagittis iaculis faucibus. Nam sagittis,
+              <td class="notTD">1.05</td>
-            mauris ut vestibulum tincidunt, dui ante sagittis ex, id finibus
+             </tr>
-             eros augue eu velit. Maecenas vulputate ante sed hendrerit
+             <tr class="notTR">
-             vestibulum. Lorem ipsum dolor sit amet, consectetur adipiscing elit.
+              <td class="notTD">Sample pooled (5ng)</td>
-             Nam enim purus, porta in leo id, accumsan gravida augue. Vestibulum
+              <td class="notTD">1028.5)</td>
-            quis sollicitudin lectus, a pretium erat. Donec hendrerit imperdiet
+              <td class="notTD">1.61</td>
-            turpis, vel convallis est pharetra quis. Curabitur molestie
+              <td class="notTD">1.05</td>
-             porttitor turpis, in fringilla arcu lacinia et. </p>
+             </tr>
+             <tr class="notTR">
+              <td class="notTD">Sample pooled (1ng)</td>
+              <td class="notTD">1028.7)</td>
+              <td class="notTD">1.61</td>
+              <td class="notTD">1.04</td>
+             </tr>
+          </tbody>
+        </table>
+        <p class="date">08.20.10</p>
+        <p class="p3N"> Digestion - 30 min, 5 U, from 5 to 1 µg with control
+          without enzyme from PCR 7 </p>
+        <p class="p2N">A new digestion was performed with different quantity of
+          DNA to digest for each sample. An undigested sample from the PCR 7 was
+          used as a negative control.</p>
+        <p class="p3N">Gel electrophoresis 10 - Digestion conditions comparison
+        </p>
+        <p class="p2N">A gel with 4% of agarose was made to compare each sample.
+          The staining was performed with GelRed because it is more specific of
+          single-strand DNA than ethidium bromide and SYBR Green.</p>
+        <div><img src="https://2019.igem.org/wiki/images/thumb/4/43/T--Pasteur_Paris--Week3.png/800px-T--Pasteur_Paris--Week3.png"
+             style="width:70%; margin-left:10%"> </div>
+        <p class="date"> 08.21.19 </p>
+        <p class="p3N"> PCR 8 - Generation of new materials </p>
+        <p class="p2N">A new PCR was performed to produce starting material. <br>
+          Samples were analyzed using a Nanodrop 2000 spectrophotometer.</p>
+        <table class="notTABLE">
+          <tbody>
+             <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration(ng/µl</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ratio</td>
+             </tr>
+             <tr class="notTR">
+              <td class="notTD">PCR 8 pooled</td>
+              <td class="notTD">1093.9</td>
+              <td class="notTD">1.63</td>
+              <td class="notTD">1.11</td>
+             </tr>
+          </tbody>
+        </table>
          </div>
        </div>
@@ Line 1,467: / Line 2,666: @@
            </div>
          </div>
-         <div id="AU4" class="ent" style="overflow-x:hidden;overflow:scroll;width:85%;margin-left:7.5%;opacity:0;transition:0.3s;">
+         <div id="AU4" class="ent"  >
-           <p style="color:black;font-family:roboto;font-size:20px;font-weight:100;text-align:justify;margin-right:5%;">
+                  <p class="p1N"> August Week 4 &nbsp;: &nbsp; 08/26 &nbsp;to
-            Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
+          &nbsp;09/1 </p>
-            purus augue, dictum non molestie eget, posuere a mauris. Donec
+        <br>
-             fringilla eros in ipsum molestie eleifend. Maecenas viverra ex sed
+        <br>
-             dolor blandit, at finibus orci rhoncus. Aliquam non dui est. In
+        <br>
-             mattis tincidunt nisl, at egestas felis dignissim id. Nam
+        <br>
-            condimentum accumsan tellus quis ornare. Vestibulum quis augue
+        <p class="date"> 08.26.19 </p>
-            malesuada, tempor lorem ut, accumsan purus. Integer nibh nulla,
+        <br>
-            malesuada ut sodales non, blandit non ligula. Donec venenatis
+        <p class="p3N"> Purification of PCR product &nbsp;-&nbsp; illustra
-             faucibus pulvinar. Sed tincidunt ultrices eros, nec cursus dui
+          MicroSpin G-25 Columns kit from GE Healthcare &nbsp;-&nbsp; PCR 9 </p>
-             molestie nec. Integer ac molestie lacus. Suspendisse lacinia mauris
+        <p class="p2N"> The PCR 9 products were purified according to the
-            in dolor ullamcorper sodales. Lorem ipsum dolor sit amet,
+          manufacturer’s protocol. Then, samples were analyzed using a Nanodrop
-             consectetur adipiscing elit. Pellentesque ut elit vitae neque luctus
+spectrophotometer. </p>
-             pellentesque porta sit amet orci.
+        <br>
-            Sed eget ante ipsum. Morbi pharetra lacinia purus, vitae efficitur
+        <table class="notTABLE">
-            eros convallis vitae. Aenean in consequat tortor, vitae dapibus
+          <tbody>
-             nisi. Nunc non imperdiet tortor. Curabitur aliquet facilisis tempus.
+            <tr class="NotTR">
-             Integer viverra orci sit amet erat fringilla, sed interdum diam
+              <td class="notTD">Sample</td>
-             euismod. Ut ac vulputate orci. Nunc iaculis tellus quis massa
+              <td class="notTD">Concentration (ng/μL)</td>
-             convallis semper. Nunc nec consectetur arcu. In lectus lectus,
+              <td class="notTD">260/280 ratio</td>
-            tempor id scelerisque vitae, egestas at purus. Curabitur vel erat
+              <td class="notTD">260/230 ratio</td>
-            quis dui pretium lobortis nec a tellus.
+            </tr>
-            Duis at efficitur sem. Suspendisse potenti. Donec pretium egestas
+            <tr class="notTR">
-            suscipit. Vivamus in vulputate urna. Etiam ullamcorper aliquam
+              <td class="notTD">PCR 9 purified by column</td>
-            blandit. Phasellus accumsan volutpat tortor non dignissim. Nunc
+              <td class="notTD">868.7</td>
-            vestibulum tellus vel ex pulvinar convallis. Etiam ultricies
+              <td class="notTD">1.58</td>
-             convallis malesuada. Praesent sed cursus lorem, vitae iaculis ipsum.
+              <td class="notTD">1.03</td>
-             Vestibulum risus risus, vulputate non metus non, posuere finibus
+            </tr>
-             magna. Nulla hendrerit lorem ac lacus ornare porta. Vivamus ligula
+          </tbody>
-            lorem, ullamcorper eu nisl eget, bibendum condimentum dui.
+        </table>
-             Aenean consectetur tincidunt ullamcorper. Vestibulum tempus, mi nec
+        <br>
-             gravida euismod, massa est consectetur ligula, vitae mattis nunc
+        <p class="p3N"> Digestion &nbsp;-&nbsp; from PCR 9 </p>
-             mauris vel tortor. Vivamus ac mauris ac odio mollis volutpat vitae
+        <p class="p2N"> After the purification, the sample was digested
-            ac felis. Proin eu ex et erat commodo vulputate. Morbi quis velit
+          according to the manufacturer’s instructions with 4 µg of DNA. </p>
-             eleifend libero finibus hendrerit. Mauris egestas ultrices odio in
+        <br>
-             rutrum. Pellentesque ut laoreet purus. In vitae orci ac tellus
+        <p class="p3N"> Phenol/chloroform extraction &nbsp;-&nbsp; Digested
-            eleifend condimentum. Phasellus at purus quis orci tempor convallis
+          products from PCR 9 </p>
-             vitae non nibh.
+        <p class="p2N"> The sample was purified by phenol/chloroform according
-            Cras hendrerit interdum lectus vel dictum. Donec nec lectus mattis,
+          to the protocol previously described. Then, samples were analyzed
-            aliquam tortor vitae, convallis sem. Suspendisse vitae dui tortor.
+          using a Nanodrop 2000 spectrophotometer. </p>
-            Lorem ipsum dolor sit amet, consectetur adipiscing elit. Suspendisse
+        <br>
-            egestas, lacus a lacinia molestie, leo nibh malesuada velit, sed
+        <br>
-            iaculis nulla libero nec dui. Maecenas fringilla, massa ut pretium
+        <table class="notTABLE">
-             hendrerit, lorem urna tristique ligula, sed pharetra eros diam vel
+          <tbody>
-             odio. Aenean consectetur eu massa id volutpat. Nunc vel ex laoreet,
+             <tr class="NotTR">
-             aliquam augue ac, ullamcorper nunc. Pellentesque ut tempor dolor, ac
+              <td class="notTD">Sample</td>
-             eleifend ante.
+              <td class="notTD">Concentration (ng/μL)</td>
-             Pellentesque accumsan mauris in ante venenatis, nec elementum enim
+              <td class="notTD">260/280 ratio</td>
-             ornare. In varius rutrum odio imperdiet ultricies. Pellentesque nec
+              <td class="notTD">260/230 ration</td>
-             feugiat tortor. Donec euismod leo orci, quis sodales felis placerat
+             </tr>
-             ac. Donec a nibh dapibus, vulputate elit non, pretium nisl. Sed at
+            <tr class="notTR">
-             est massa. Fusce at semper tortor. In cursus lorem lectus, nec
+              <td class="notTD">PCR 9 purified by phenol/chloroform</td>
-             suscipit nibh euismod eu. Morbi malesuada, lacus ut pellentesque
+              <td class="notTD">17</td>
-             semper, diam tellus vulputate lectus, et varius ligula velit vitae
+              <td class="notTD">1.48</td>
-             elit. Praesent gravida, ex sit amet ornare bibendum, metus turpis
+              <td class="notTD">1.34</td>
-             maximus ipsum, eget interdum elit urna eu quam. Fusce tempor eu elit
+             </tr>
-             id efficitur. Curabitur vitae lorem vel arcu posuere blandit at et
+          </tbody>
-             dolor. Nunc posuere pharetra odio vitae iaculis. In blandit dolor
+        </table>
-             quis magna cursus aliquet. Aliquam luctus hendrerit mauris ut
+        <br>
-             tristique. Vivamus pharetra scelerisque nisl vel consequat.
+        <p class="p3N"> Interpretation </p>
-             Integer iaculis enim et dui cursus lacinia. Fusce nec ultricies
+        <p class="p2N"> We lose a huge quantity of DNA. Thus, the concentration
-             quam, quis molestie augue. Cras mauris quam, eleifend quis sagittis
+          is too low to begin the first round of SELEX. </p>
-             eu, venenatis non mauris. Nulla sollicitudin ut nisl at ultrices.
+        <br>
-             Vivamus fermentum placerat purus et gravida. Integer vulputate quam
+        <p class="date"> 08.27.19 </p>
-             nec nunc porttitor, vel gravida est aliquam. Aenean elementum eget
+        <br>
-             velit ut tincidunt. Fusce sollicitudin, magna vitae sodales
+        <p class="p3N"> Digestion - from PCR 9 </p>
-             tincidunt, diam turpis fermentum urna, eget maximus est libero
+        <p class="p2N"> We purify the digested samples using either 2.5 X volume
-             lacinia nibh. Donec sagittis elementum molestie. Donec suscipit
+          of ethanol, as previously, or 1 X volume of isopropanol. The samples
-             maximus laoreet. Proin sed convallis enim. Nullam dignissim pharetra
+          were then analyzed using a Nanodrop 2000 spectrophotometer. </p>
-             dapibus. Aenean vel varius ipsum.
+        <br>
-             Ut eget rutrum nunc, sit amet tempor arcu. Etiam a tempus magna.
+        <table class="notTABLE">
-             Vivamus leo nunc, consectetur non sagittis euismod, eleifend in
+          <tbody>
-             ante. Etiam imperdiet at dui vitae lobortis. Donec at mattis ipsum.
+             <tr class="NotTR">
-             Nam ut ante condimentum, sodales mauris vel, tempor massa. Curabitur
+              <td class="notTD">Sample</td>
-             feugiat libero consectetur velit viverra, nec fermentum leo
+              <td class="notTD">Concentration (ng/μL)</td>
-             ultrices. Nulla laoreet in orci consequat condimentum. Donec et
+              <td class="notTD">260/280 ratio</td>
-             posuere mauris, sagittis eleifend augue.
+              <td class="notTD">260/230 ration</td>
-             Nunc venenatis enim in auctor rutrum. Nullam molestie arcu quis
+             </tr>
-             turpis mollis mattis. Phasellus elementum lectus vitae odio
+            <tr class="notTR">
-             tincidunt, vitae congue nisl volutpat. Fusce sodales fringilla neque
+              <td class="notTD">PCR 9 purified with ethanol &nbsp; &nbsp;1</td>
-             sed posuere. Morbi risus sem, imperdiet iaculis neque ac, malesuada
+              <td class="notTD">9.2</td>
-             porta lectus. Quisque congue vel lorem vel commodo. In sit amet
+              <td class="notTD">1.6</td>
-             gravida purus. Aliquam erat volutpat.
+              <td class="notTD">0.91</td>
-             Praesent pellentesque pharetra sem sed condimentum. Quisque a
+             </tr>
-             bibendum turpis, et malesuada ipsum. Nunc viverra quam in magna
+             <tr class="notTR">
-             viverra facilisis. Ut pharetra eget urna sit amet luctus. Phasellus
+              <td class="notTD">PCR 9 purified with ethanol &nbsp; &nbsp;2</td>
-             condimentum fermentum tortor, ut venenatis augue suscipit non.
+              <td class="notTD">7.3</td>
-             Maecenas facilisis arcu non diam vehicula lobortis. Suspendisse ut
+              <td class="notTD">1.5</td>
-             neque libero. Quisque sagittis iaculis faucibus. Nam sagittis,
+              <td class="notTD">0.91</td>
-             mauris ut vestibulum tincidunt, dui ante sagittis ex, id finibus
+             </tr>
-             eros augue eu velit. Maecenas vulputate ante sed hendrerit
+            <tr class="notTR">
-             vestibulum. Lorem ipsum dolor sit amet, consectetur adipiscing elit.
+              <td class="notTD">PCR 9 purified with isopropanol &nbsp; &nbsp;1</td>
-             Nam enim purus, porta in leo id, accumsan gravida augue. Vestibulum
+              <td class="notTD">44.9</td>
-             quis sollicitudin lectus, a pretium erat. Donec hendrerit imperdiet
+              <td class="notTD">1.41</td>
-             turpis, vel convallis est pharetra quis. Curabitur molestie
+              <td class="notTD">1.10</td>
-             porttitor turpis, in fringilla arcu lacinia et. </p>
+             </tr>
+             <tr class="notTR">
+              <td class="notTD">PCR 9 purified with isopropanol &nbsp; &nbsp;1</td>
+              <td class="notTD">48.1</td>
+              <td class="notTD">1.42</td>
+              <td class="notTD">1.39</td>
+             </tr>
+          </tbody>
+        </table>
+        <p class="p3N"> Interpretations </p>
+        <p class="p2N"> The concentration of DNA are much higher when we used
+          isopropanol compared to ethanol. </p>
+        <br>
+        <p class="p3N"> Conclusion </p>
+        <p class="p2N"> Isopropanol is used for the next purifications by
+          phenol/chloroform. These concentrations are enough to perform the
+          first round of SELEX on one bacteria. However, we want to select
+          aptamers for two different bacteria so we must perform this step
+          again. </p>
+        <br>
+        <p class="date"> 08.28.19 </p>
+        <p class="p3N"> Bacteria culture &nbsp;-&nbsp; E. faecium and S. aureus
+        </p>
+        <p class="p2N"> The strains E. faecium and S. aureus were grown on Petri
+          dish on Blood agar and LB media respectively. The plates were
+          incubated at 37°C during 24 hours. </p>
+        <div> <img src="https://2019.igem.org/wiki/images/c/c5/T--Pasteur_Paris--NotebookImg13.jpg"
+             style="width:70%;margin-left:10%"> </div>
+        <p class="p3N"> Digestion &nbsp;-&nbsp; from PCR9 </p>
+        <p class="p2N"> The PCR 9 products were digested using 4 µg of DNA
+          according to the manufacturer’s protocol. </p>
+        <p class="p3N"> Phenol/chloroform extraction with Isopropanol
+          &nbsp;-&nbsp; Digested products from PCR 9 </p>
+        <p class="p2N"> After digestion, the samples were purified using 1 X
+          volume of isopropanol. The samples are then analysed using a Nanodrop
+spectrophotometer. </p>
+        <br>
+        <table class="notTABLE">
+          <tbody>
+             <tr class="NotTR">
+              <td class="notTD">Sample</td>
+              <td class="notTD">Concentration (ng/μL)</td>
+              <td class="notTD">260/280 ratio</td>
+              <td class="notTD">260/230 ration</td>
+             </tr>
+            <tr class="notTR">
+              <td class="notTD">PCR 9 purified by phenol/chloroform &nbsp;
+                &nbsp;1</td>
+              <td class="notTD">42.1</td>
+              <td class="notTD">1.46</td>
+              <td class="notTD">1.40</td>
+             </tr>
+            <tr class="notTR">
+              <td class="notTD">PCR 9 purified by phenol/chloroform 2</td>
+              <td class="notTD">3.1</td>
+              <td class="notTD">1.65</td>
+              <td class="notTD">0.71</td>
+             </tr>
+             <tr class="notTR">
+              <td class="notTD">PCR 9 purified by phenol/chloroform 3</td>
+              <td class="notTD">3.1</td>
+              <td class="notTD">1.55</td>
+              <td class="notTD">1.64</td>
+             </tr>
+             <tr class="notTR">
+              <td class="notTD">PCR 9 purified by phenol/chloroform 4</td>
+              <td class="notTD">8.6</td>
+              <td class="notTD">1.43</td>
+              <td class="notTD">1.19</td>
+             </tr>
+          </tbody>
+        </table>
+        <br>
+        <p class="p3N"> Interpretations </p>
+        <p class="p2N"> The concentration of DNA is lower than expected. We may
+          have pipeted some DNA as the pellet is invisible. </p>
+        <br>
+        <p class="p3N"> Conclusion </p>
+        <p class="p2N"> Because of these low concentrations, the first round of
+          SELEX cannot be performed. </p>
+        <br>
+        <p class="date"> 08.29.19 </p>
+        <br>
+        <p class="p3N"> Bacteria culture &nbsp;-&nbsp; E. faecium and S. aureus
+        </p>
+        <p class="p2N"> Few colonies of E. faecium and S. aureus were taken from
+          the Petri dish culture of the 28.08 and grow in their respective
+          liquid media at 37°C under agitation. After 3 hours of growth, they
+          are centrifuged and the pellet of bacteria is resuspended in PBS 1X.
+          Dilutions were performed until the OD reached between 0.5 and 0.6. </p>
+        <br>
+        <p class="p3N"> Digestion &nbsp;-&nbsp; from PCR9 </p>
+        <p class="p2N"> In order to have more DNA before starting the SELEX, we
+          performed a digestion with 4 µg of DNA from the PCR 9 samples
+          according to the manufacturer’s protocol. </p>
+        <br>
+        <p class="p3N"> Phenol/chloroform extraction with Isopropanol
+          &nbsp;-&nbsp; Digested products from PCR 9 </p>
+        <p class="p2N"> The samples were purified after digestion using 1 X
+          volume of isopropanol. Then, they were analysed with a Nanodrop 2000
+          spectrophotometer. </p>
+        <br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+             <td class="notTD">Sample</td>
+             <td class="notTD">Concentration (ng/μL)</td>
+            <td class="notTD">260/280 ratio</td>
+            <td class="notTD">260/230 ration</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">PCR 9 purified sample 1</td>
+            <td class="notTD">41.4</td>
+             <td class="notTD">1.41</td>
+             <td class="notTD">1.46</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">PCR 9 purified sample 2</td>
+             <td class="notTD">4.2</td>
+            <td class="notTD">1.25</td>
+             <td class="notTD">0.57</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">PCR 9 purified sample 3</td>
+            <td class="notTD">9.4</td>
+             <td class="notTD">1.33</td>
+             <td class="notTD">0.99</td>
+          </tr>
+           <tr class="notTR">
+             <td class="notTD">PCR 9 purified sample 4</td>
+             <td class="notTD">23.7</td>
+             <td class="notTD">1.39</td>
+             <td class="notTD">1.08</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">PCR 9 purified sample 5</td>
+            <td class="notTD">11.1</td>
+             <td class="notTD">1.37</td>
+             <td class="notTD">0.41</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">PCR 9 purified sample 6</td>
+            <td class="notTD">27.8</td>
+             <td class="notTD">1.40</td>
+             <td class="notTD">0.90</td>
+          </tr>
+        </table>
+        <br>
+        <p class="p3N">
+          Conclusion
+        </p>
+        <br>
+        <p class="p2N">
+          After the purification, we have enough DNA concentration to start the SELEX on the two bacteria.
+        </p>
+        <p class="p3N">
+          SELEX 1 Round 1
+        </p>
+        <p class="p2N">
+          The first round of SELEX was performed  with 2 hours of incubation. The samples are then analyzed using a Nanodrop 2000 spectrophotometer.
+        </p>
+        <br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+             <td class="notTD">Sample</td>
+             <td class="notTD">Concentration (ng/μL)</td>
+             <td class="notTD">260/280 ratio</td>
+             <td class="notTD">260/230 ration</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">S. aureus</td>
+            <td class="notTD">60.1</td>
+             <td class="notTD">1.61</td>
+            <td class="notTD">0.96</td>
+          </tr>
+           <tr class="notTR">
+             <td class="notTD">E. faecium</td>
+             <td class="notTD">43.6</td>
+             <td class="notTD">1.69</td>
+             <td class="notTD">1.09</td>
+          </tr>
+        </table>
+        <br>
+        <p class="p3N">
+          PCR 10 &nbsp;-&nbsp; Amplification of EFr1 & SAr1
+        </p>
+        <p class="p2N">
+          This PCR was performed to amplified the DNA after the first round of SELEX. We tried two different concentrations for both samples. The PCR followed the same program as described previously. It was run overnight and hold at 4°C.
+        </p>
+        <br>
+        <table class="notTABLE" style="border-color:transparent;">
+          <tr class="notTR">
+             <td></td>
+             <td class="notTD" colspan="2">S aureus</td>
+          </tr>
+          <tr class="NotTR">
+             <td class="notTD">Components</td>
+            <td class="notTD">SA 1 (1/10)</td>
+            <td class="notTD">SA 2 (1/90)</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Phusion buffer</td>
+            <td class="notTD">10</td>
+            <td class="notTD">10</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">dNTPs</td>
+            <td class="notTD">1</td>
+            <td class="notTD">1</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Forward primer</td>
+            <td class="notTD">2</td>
+            <td class="notTD">2</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Reverse primer</td>
+            <td class="notTD">2</td>
+            <td class="notTD">2</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Template DNA (1/10)</td>
+            <td class="notTD">1</td>
+            <td class="notTD">0</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Template DNA (1/90)</td>
+            <td class="notTD">0</td>
+            <td class="notTD">1</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Phusion DNA polymerase</td>
+            <td class="notTD">0.5</td>
+            <td class="notTD">0.5</td>
+          </tr>
+           <tr class="notTR">
+             <td class="notTD">Nulease-free water</td>
+            <td class="notTD">33.5</td>
+             <td class="notTD">33.5</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Total volume</td>
+            <td class="notTD">50</td>
+            <td class="notTD">50</td>
+          </tr>
+        </table>
+        <br><br><br>
+        <table class="notTABLE" style="border-color:transparent;">
+          <tr class="notTR">
+            <td></td>
+            <td class="notTD" colspan="2">E. faecium</td>
+          </tr>
+          <tr class="NotTR">
+             <td class="notTD">Components</td>
+            <td class="notTD">SA 1 (1/10)</td>
+            <td class="notTD">SA 2 (1/80)</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Phusion buffer</td>
+            <td class="notTD">10</td>
+            <td class="notTD">10</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">dNTPs</td>
+            <td class="notTD">1</td>
+            <td class="notTD">1</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Forward primer</td>
+            <td class="notTD">2</td>
+            <td class="notTD">2</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Reverse primer</td>
+            <td class="notTD">2</td>
+            <td class="notTD">2</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Template DNA (1/10)</td>
+            <td class="notTD">1</td>
+            <td class="notTD">0</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Template DNA (1/80)</td>
+            <td class="notTD">0</td>
+            <td class="notTD">1</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Phusion DNA polymerase</td>
+            <td class="notTD">0.5</td>
+             <td class="notTD">0.5</td>
+          </tr>
+           <tr class="notTR">
+             <td class="notTD">Nulease-free water</td>
+            <td class="notTD">33.5</td>
+            <td class="notTD">33.5</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Total volume</td>
+            <td class="notTD">50</td>
+            <td class="notTD">50</td>
+          </tr>
+        </table>
+        <br>
+        <p class="p2N">
+          The samples are analyzed using a Nanodrop 2000 spectrophotometer.
+        </p>
+        <br><br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+             <td class="notTD">Sample</td>
+            <td class="notTD">Concentration (ng/μL)</td>
+            <td class="notTD">260/280 ratio</td>
+            <td class="notTD">260/230 ration</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">PCR 10 - Round 1 - SA &nbsp;&nbsp; 1</td>
+            <td class="notTD">1253.9</td>
+             <td class="notTD">1.62</td>
+             <td class="notTD">1.05</td>
+          </tr>
+           <tr class="notTR">
+             <td class="notTD">PCR 10 - Round 1 - SA &nbsp;&nbsp; 2</td>
+            <td class="notTD">1200.4</td>
+             <td class="notTD">1.64</td>
+             <td class="notTD">1.09</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">PCR 10 - Round 1 - EF &nbsp;&nbsp; 1</td>
+             <td class="notTD">1118.9</td>
+             <td class="notTD">1.56</td>
+             <td class="notTD">0.89</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">PCR 10 - Round 1 - EF &nbsp;&nbsp; 2</td>
+            <td class="notTD">1207.9</td>
+             <td class="notTD">1.66</td>
+             <td class="notTD">1.13</td>
+          </tr>
+        </table>
+        <br>
+        <p class="date">
+.30.19
+        </p>
+        <br>
+        <p class="p3N">
+          Gel electrophoresis 12 &nbsp;-&nbsp; PCR 10
+        </p>
+        <p class="p2N">
+          On a 4% agarose gel, samples before and after the PCR 12 were run.
+        </p>
          </div>
        </div>
@@ Line 1,573: / Line 3,123: @@
            </div>
          </div>
-         <div id="AU5" class="ent" style="overflow-x:hidden;overflow:scroll;width:85%;margin-left:7.5%;opacity:0;transition:0.3s;">
+         <div id="AU5" class="ent"  >
             <p style="color:black;font-family:roboto;font-size:20px;font-weight:100;text-align:justify;margin-right:5%;">
              Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
@@ Line 1,679: / Line 3,229: @@
            </div>
          </div>
-         <div id="SE1" class="ent" style="overflow-x:hidden;overflow:scroll;width:85%;margin-left:7.5%;opacity:0;transition:0.3s;">
+         <div id="SE1" class="ent"  >
-           <p style="color:black;font-family:roboto;font-size:20px;font-weight:100;text-align:justify;margin-right:5%;">
+                   <p class="p1N">
-             Lorem ipsum dolor sit amet, consectetur adipiscing elit. Praesent
+          September Week 1 &nbsp;: &nbsp;09/05 &nbsp;to &nbsp;09/15
-             purus augue, dictum non molestie eget, posuere a mauris. Donec
+        </p>
-             fringilla eros in ipsum molestie eleifend. Maecenas viverra ex sed
+        <br>
-             dolor blandit, at finibus orci rhoncus. Aliquam non dui est. In
+        <p class="p3N">
-             mattis tincidunt nisl, at egestas felis dignissim id. Nam
+          Digestion  &nbsp;-&nbsp; EFr1 &amp; SAr1
-             condimentum accumsan tellus quis ornare. Vestibulum quis augue
+        </p>
-             malesuada, tempor lorem ut, accumsan purus. Integer nibh nulla,
+        <p class="p2N">
-             malesuada ut sodales non, blandit non ligula. Donec venenatis
+          The digestion was performed with 4 µg of DNA according to the manufacturer’s protocol.
-             faucibus pulvinar. Sed tincidunt ultrices eros, nec cursus dui
+        </p>
-            molestie nec. Integer ac molestie lacus. Suspendisse lacinia mauris
+        <br>
-            in dolor ullamcorper sodales. Lorem ipsum dolor sit amet,
+        <table class="notTABLE">
-             consectetur adipiscing elit. Pellentesque ut elit vitae neque luctus
+          <tr class="NotTR">
-             pellentesque porta sit amet orci.
+            <td class="notTD">Sample</td>
-             Sed eget ante ipsum. Morbi pharetra lacinia purus, vitae efficitur
+            <td class="notTD">Concentration (ng/&mu;L)</td>
-             eros convallis vitae. Aenean in consequat tortor, vitae dapibus
+            <td class="notTD">260/280 ratio</td>
-             nisi. Nunc non imperdiet tortor. Curabitur aliquet facilisis tempus.
+             <td class="notTD">260/230 ratio</td>
-             Integer viverra orci sit amet erat fringilla, sed interdum diam
+          </tr>
-             euismod. Ut ac vulputate orci. Nunc iaculis tellus quis massa
+          <tr class="notTR">
-             convallis semper. Nunc nec consectetur arcu. In lectus lectus,
+             <td class="notTD">EFr1 digested</td>
-             tempor id scelerisque vitae, egestas at purus. Curabitur vel erat
+             <td class="notTD">27.4</td>
-             quis dui pretium lobortis nec a tellus.
+             <td class="notTD">1.87</td>
-             Duis at efficitur sem. Suspendisse potenti. Donec pretium egestas
+             <td class="notTD">0.26</td>
-             suscipit. Vivamus in vulputate urna. Etiam ullamcorper aliquam
+          </tr>
-             blandit. Phasellus accumsan volutpat tortor non dignissim. Nunc
+           <tr class="notTR">
-             vestibulum tellus vel ex pulvinar convallis. Etiam ultricies
+             <td class="notTD">SAr1 digested</td>
-             convallis malesuada. Praesent sed cursus lorem, vitae iaculis ipsum.
+             <td class="notTD">30.2</td>
-             Vestibulum risus risus, vulputate non metus non, posuere finibus
+             <td class="notTD">1.89</td>
-             magna. Nulla hendrerit lorem ac lacus ornare porta. Vivamus ligula
+             <td class="notTD">0.28</td>
-             lorem, ullamcorper eu nisl eget, bibendum condimentum dui.
+          </tr>
-             Aenean consectetur tincidunt ullamcorper. Vestibulum tempus, mi nec
+        </table>
-             gravida euismod, massa est consectetur ligula, vitae mattis nunc
+        <br>
-             mauris vel tortor. Vivamus ac mauris ac odio mollis volutpat vitae
+        <p class="p3N">
-             ac felis. Proin eu ex et erat commodo vulputate. Morbi quis velit
+        Purification of digested products EFr1 &amp; SAr1 &nbsp;-&nbsp; kit Monarch from NEB
-             eleifend libero finibus hendrerit. Mauris egestas ultrices odio in
+        </p>
-             rutrum. Pellentesque ut laoreet purus. In vitae orci ac tellus
+        <p class="p2N">
-             eleifend condimentum. Phasellus at purus quis orci tempor convallis
+          A new kit of purification was tried to purify the PCR Products. The experiment was performed according to the manufacturer’s recommendations.
-             vitae non nibh.
+        </p>
-             Cras hendrerit interdum lectus vel dictum. Donec nec lectus mattis,
+        <br>
-            aliquam tortor vitae, convallis sem. Suspendisse vitae dui tortor.
+        <p class="p2N">
-            Lorem ipsum dolor sit amet, consectetur adipiscing elit. Suspendisse
+          The samples are checked using a Nanodrop 2000 spectrophotometer.
-            egestas, lacus a lacinia molestie, leo nibh malesuada velit, sed
+        </p>
-             iaculis nulla libero nec dui. Maecenas fringilla, massa ut pretium
+        <br>
-             hendrerit, lorem urna tristique ligula, sed pharetra eros diam vel
+        <table class="notTABLE">
-             odio. Aenean consectetur eu massa id volutpat. Nunc vel ex laoreet,
+          <tr class="NotTR">
-             aliquam augue ac, ullamcorper nunc. Pellentesque ut tempor dolor, ac
+             <td class="notTD">Sample</td>
-             eleifend ante.
+             <td class="notTD">Concentration (ng/µl)</td>
-             Pellentesque accumsan mauris in ante venenatis, nec elementum enim
+             <td class="notTD">260/280 ratio</td>
-             ornare. In varius rutrum odio imperdiet ultricies. Pellentesque nec
+            <td class="notTD">260/230 ratio</td>
-             feugiat tortor. Donec euismod leo orci, quis sodales felis placerat
+          </tr>
-             ac. Donec a nibh dapibus, vulputate elit non, pretium nisl. Sed at
+          <tr class="notTR">
-            est massa. Fusce at semper tortor. In cursus lorem lectus, nec
+            <td class="notTD">EFr1 purified</td>
-            suscipit nibh euismod eu. Morbi malesuada, lacus ut pellentesque
+            <td class="notTD">7.8</td>
-             semper, diam tellus vulputate lectus, et varius ligula velit vitae
+             <td class="notTD">1.27</td>
-             elit. Praesent gravida, ex sit amet ornare bibendum, metus turpis
+             <td class="notTD">0.27</td>
-             maximus ipsum, eget interdum elit urna eu quam. Fusce tempor eu elit
+          </tr>
-             id efficitur. Curabitur vitae lorem vel arcu posuere blandit at et
+          <tr class="notTR">
-             dolor. Nunc posuere pharetra odio vitae iaculis. In blandit dolor
+             <td class="notTD">EFr1 wash 1</td>
-             quis magna cursus aliquet. Aliquam luctus hendrerit mauris ut
+             <td class="notTD">-1.7</td>
-             tristique. Vivamus pharetra scelerisque nisl vel consequat.
+             <td class="notTD">0.58</td>
-             Integer iaculis enim et dui cursus lacinia. Fusce nec ultricies
+             <td class="notTD">-0.03</td>
-             quam, quis molestie augue. Cras mauris quam, eleifend quis sagittis
+          </tr>
-             eu, venenatis non mauris. Nulla sollicitudin ut nisl at ultrices.
+          <tr class="notTR">
-             Vivamus fermentum placerat purus et gravida. Integer vulputate quam
+             <td class="notTD">EFr1 wash 2</td>
-             nec nunc porttitor, vel gravida est aliquam. Aenean elementum eget
+             <td class="notTD">2.5</td>
-             velit ut tincidunt. Fusce sollicitudin, magna vitae sodales
+            <td class="notTD">5.51</td>
-             tincidunt, diam turpis fermentum urna, eget maximus est libero
+             <td class="notTD">0.27</td>
-             lacinia nibh. Donec sagittis elementum molestie. Donec suscipit
+          </tr>
-             maximus laoreet. Proin sed convallis enim. Nullam dignissim pharetra
+          <tr class="notTR">
-             dapibus. Aenean vel varius ipsum.
+             <td class="notTD">EFr1 purified</td>
-             Ut eget rutrum nunc, sit amet tempor arcu. Etiam a tempus magna.
+            <td class="notTD">7.8</td>
-             Vivamus leo nunc, consectetur non sagittis euismod, eleifend in
+             <td class="notTD">1.27</td>
-             ante. Etiam imperdiet at dui vitae lobortis. Donec at mattis ipsum.
+             <td class="notTD">0.27</td>
-             Nam ut ante condimentum, sodales mauris vel, tempor massa. Curabitur
+          </tr>
-             feugiat libero consectetur velit viverra, nec fermentum leo
+          <tr class="notTR">
-             ultrices. Nulla laoreet in orci consequat condimentum. Donec et
+             <td class="notTD">SAr1 purified</td>
-             posuere mauris, sagittis eleifend augue.
+             <td class="notTD">7.2</td>
-             Nunc venenatis enim in auctor rutrum. Nullam molestie arcu quis
+             <td class="notTD">1.52</td>
-             turpis mollis mattis. Phasellus elementum lectus vitae odio
+             <td class="notTD">0.58</td>
-             tincidunt, vitae congue nisl volutpat. Fusce sodales fringilla neque
+          </tr>
-             sed posuere. Morbi risus sem, imperdiet iaculis neque ac, malesuada
+          <tr class="notTR">
-             porta lectus. Quisque congue vel lorem vel commodo. In sit amet
+             <td class="notTD">SAr1 wash 1</td>
-             gravida purus. Aliquam erat volutpat.
+             <td class="notTD">22.5</td>
-             Praesent pellentesque pharetra sem sed condimentum. Quisque a
+             <td class="notTD">1.29</td>
-             bibendum turpis, et malesuada ipsum. Nunc viverra quam in magna
+             <td class="notTD">0.53</td>
-             viverra facilisis. Ut pharetra eget urna sit amet luctus. Phasellus
+          </tr>
-             condimentum fermentum tortor, ut venenatis augue suscipit non.
+          <tr class="notTR">
-             Maecenas facilisis arcu non diam vehicula lobortis. Suspendisse ut
+             <td class="notTD">SAr1 wash 2</td>
-             neque libero. Quisque sagittis iaculis faucibus. Nam sagittis,
+             <td class="notTD">2.4</td>
-             mauris ut vestibulum tincidunt, dui ante sagittis ex, id finibus
+             <td class="notTD">8.31</td>
-             eros augue eu velit. Maecenas vulputate ante sed hendrerit
+             <td class="notTD">0.30</td>
-             vestibulum. Lorem ipsum dolor sit amet, consectetur adipiscing elit.
+          </tr>
-             Nam enim purus, porta in leo id, accumsan gravida augue. Vestibulum
+        </table>
-             quis sollicitudin lectus, a pretium erat. Donec hendrerit imperdiet
+        <br>
-             turpis, vel convallis est pharetra quis. Curabitur molestie
+        <p class="p3N">
-             porttitor turpis, in fringilla arcu lacinia et. </p>
+          Interpretations
+        </p>
+        <p class="p2N">
+          The concentrations are really low. Moreover, the quantity of DNA within the sample is not high because the ratio 260/280 is not close to 2. Finally, the ratio 260/230 did not change increase before and after the purification.
+        </p>
+        <br>
+        <p class="p3N">
+          Conclusion
+        </p>
+        <p class="p2N">
+          This kit cannot be used to purify our DNA after digestion.
+        </p>
+        <br>
+        <p class="p3N">
+          Purification of digested products EFr1 &amp; SAr1 &nbsp;-&nbsp;  illustra MicroSpin G-25 Columns kit from GE Healthcare
+        </p>
+        <br>
+        <p class="p2N">
+          The purification was performed according to the manufacturer’s protocol.
+The samples are checked using a Nanodrop 2000 spectrophotometer.
+        </p>
+        <br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+          	<td class="notTD">Sample</td>
+             <td class="notTD">Concentration (ng/&mu;L)</td>
+             <td class="notTD">260/280 ratio</td>
+             <td class="notTD">260/230 ratio</td>
+          </tr>
+          <tr class="notTR">
+          	<td class="notTD">EFr1 purified</td>
+             <td class="notTD">53.2</td>
+             <td class="notTD">1.62</td>
+             <td class="notTD">0.24</td>
+          </tr>
+          <tr class="notTR">
+          	<td class="notTD">SAr1 purified</td>
+             <td class="notTD">56.2</td>
+             <td class="notTD">1.54</td>
+             <td class="notTD">0.27</td>
+          </tr>
+        </table>
+        <br>
+        <p class="p3N">
+          Interpretation
+        </p>
+        <p class="p2N">
+          Even if the concentrations are quite elevated, we noticed that the ratio 260/230 did not change before and after the purification using these columns. Hence, the enzymes may not be  purified by this technique.
+        </p>
+        <br>
+        <p class="p3N">
+          Conclusion
+        </p>
+        <p class="p2N">
+          We tried several kits to purify our PCR products but none of them worked. Thus, we decided to directly continue with the digestion after the PCR without purifying. All the enzymes will be removed during the phenol/chloroform extraction just before the SELEX.
+        </p>
+        <br>
+        <br>
+        <p class="date">
+.06.19
+        </p>
+        <br>
+        <p class="p3N">
+          Bacteria culture &nbsp;-&nbsp; E. faecium and S. aureus
+        </p>
+        <p class="p2N">
+          Few colonies of E. faecium and S. aureus were taken from the Petri dish culture of the 28.08 and grow in their respective liquid media at 37°C under agitation. After 3 hours of growth, they are centrifuged and the pellet of bacteria is resuspended in PBS 1X. Dilutions were performed until the OD reached between 0.5 and 0.6.
+        </p>
+        <br>
+        <p class="p3N">
+          Phenol/chloroform extraction with Isopropanol &nbsp;-&nbsp; Digested products EFr1 &amp; SAr1
+        </p>
+        <p class="p2N">
+          The samples were purified after digestion. Then, they were analysed with a Nanodrop 2000 spectrophotometer.
+        </p>
+        <br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+             <td class="notTD">Sample</td>
+             <td class="notTD">Concetration (ng/&mu;L)</td>
+            <td class="notTD">260/280 ratio</td>
+            <td class="notTD">260/230 ratio</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">EFr1 sample 1</td>
+            <td class="notTD">17.2</td>
+             <td class="notTD">1.46</td>
+             <td class="notTD">1.29</td>
+          </tr>
+           <tr class="notTR">
+             <td class="notTD">EFr1 sample 2</td>
+            <td class="notTD">26.5</td>
+             <td class="notTD">1.49</td>
+             <td class="notTD">1.44</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">EFr1 sample 3</td>
+            <td class="notTD">29.8</td>
+             <td class="notTD">1.49</td>
+             <td class="notTD">1.47</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">EFr1 sample 4</td>
+            <td class="notTD">37.2</td>
+             <td class="notTD">1.56</td>
+             <td class="notTD">1.5</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">SAr1 sample 1</td>
+             <td class="notTD">69</td>
+            <td class="notTD">1.51</td>
+            <td class="notTD">1.54</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">SAr1 sample 2</td>
+            <td class="notTD">61.5</td>
+             <td class="notTD">1.75</td>
+             <td class="notTD">0.28</td>
+          </tr>
+        </table>
+        <p class="p3N">
+          Interpretations
+        </p>
+        <p class="p2N">
+          A total quantity of 1728 pmol was obtained for E. faecium in the Round 1, and 869 pmol for <i>S. aureus</i>.
+        </p>
+        <br>
+        <p class="p3N">
+          SELEX Round 2
+        </p>
+        <p class="p2N">
+          The first round of SELEX was performed  with 2 hours of incubation. The samples are then analyzed using a Nanodrop 2000 spectrophotometer.
+        </p>
+        <br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+             <td class="notTD">Sample</td>
+             <td class="notTD">Concetration (ng/&mu;L)</td>
+             <td class="notTD">260/280 ratio</td>
+             <td class="notTD">260/230 ratio</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">SAr2</td>
+             <td class="notTD">33.8</td>
+             <td class="notTD">2.19</td>
+             <td class="notTD">1.45</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">EFr2</td>
+             <td class="notTD">32</td>
+             <td class="notTD">2.21</td>
+             <td class="notTD">1.6</td>
+          </tr>
+        </table>
+        <br><br>
+        <p class="p3N">
+          PCR 10 &nbsp;-&nbsp; Amplification of EFr2 and SAr2
+        </p>
+        <p class="p2N">
+          This PCR was performed to amplified the DNA after the second round of SELEX. The PCR followed the same program as described previously. It was run overnight and hold at 4°C.
+        </p>
+        <br><br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+             <td class="notTD">Components</td>
+             <td class="notTD">SAr1</td>
+             <td class="notTD">EFr1</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">Phusion buffer</td>
+             <td class="notTD">200</td>
+             <td class="notTD">200</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">dNTPs</td>
+            <td class="notTD">20</td>
+            <td class="notTD">20</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Forward primer</td>
+            <td class="notTD">40</td>
+            <td class="notTD">40</td>
+          </tr>
+           <tr class="notTR">
+            <td class="notTD">Reverse primer</td>
+            <td class="notTD">40</td>
+            <td class="notTD">40</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Template DNA (1/3)</td>
+            <td class="notTD">20</td>
+            <td class="notTD">20</td>
+          </tr>
+           <tr class="notTR">
+            <td class="notTD">Phusion DNA polymerase</td>
+            <td class="notTD">10</td>
+            <td class="notTD">10</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Nuclease-free water</td>
+            <td class="notTD">670</td>
+            <td class="notTD">670</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">Total volume</td>
+            <td class="notTD">1000</td>
+            <td class="notTD">1000</td>
+          </tr>
+        </table>
+        <br>
+        <p class="p2N">
+          The samples are analyzed using a Nanodrop 2000 spectrophotometer.
+        </p>
+        <br>
+        <table class="notTABLE">
+          <tr class="NotTR">
+             <td class="notTD">Sample</td>
+             <td class="notTD">Concetration (ng/&mu;L)</td>
+            <td class="notTD">260/280 ratio</td>
+            <td class="notTD">260/230 ratio</td>
+          </tr>
+          <tr class="notTR">
+            <td class="notTD">PCR 10 - Round 2 - SA</td>
+            <td class="notTD">1086.6</td>
+             <td class="notTD">1.67</td>
+             <td class="notTD">1.20</td>
+          </tr>
+          <tr class="notTR">
+             <td class="notTD">PCR 10 - Round 2 - EF</td>
+            <td class="notTD">1243.4</td>
+             <td class="notTD">1.66</td>
+             <td class="notTD">1.20</td>
+          </tr>
+        </table>
          </div>
        </div>
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Samples	Concentration (ng/µl)	260/280 ratio	260/230 ratio
DNA library	2217.65	1.89	1.56
Reverse primer	8810.9	1.95	1.58

		Volume needed (μL)
Components	Final concentration	Mix 1	Mix 2	Mix 3
Phusion HF buffer 5X	1X	50	50	50
25 mM dNTPs	0.20 mM	2	2	2
Forward primer	0.5 μM	9.75	9.75	9.75
Reverse primer	0.5 μM	10.1	10.1	10.1
25 mM Magnesium	2.5 mM	25	50	75
DNA library	10 ng	22.5	22.5	22.5
Taq DNA polymerase	0.1 U/μL	0.02	0.02	0.02
Phusion DNA polymerase	1 U/μL	0.5	0.5	0.5
Nuclease-free water	/	130.13	105.13	80.13
Total volume	/	250	250	250

A (54°C)	1.1	2.1	3.1
B	/	/	/
C (51.9°C)	1.2	2.2	3.2
D	/	/	/
E (47.6°C)	1.3	2.3	3.3
F	/	/	/
G (44.7°C)	1.4	2.4	3.4
G	/	/	/

Sample	Concentration (ng/μL)	260/280 ratio	260/230 ratio
1.1	465.9	1.39	0.66
1.2	501.7	1.42	0.69
1.3	480.4	1.43	0.68
1.4	469.9	1.43	0.65
2.1	498.3	1.40	0.68
2.2	498.7	1.43	0.70
2.3	532.9	1.42	0.71
2.4	495.2	1.42	0.69
3.1	497.8	1.43	0.70
3.2	512.2	1.43	0.70
3.3	505.5	1.42	0.70
3.4	527.2	1.41	0.70

1.	Sample 1.1 (54°C, 25 μL, Mg2+)
2.	Sample 1.2 (51.9°C, 25 μL, Mg2+)
3.	Sample 1.3 (47.6°C, 25 μL, Mg2+)
4.	Sample 1.4 (44.7°C, 25 μL, Mg2+)
5.	Sample 2.1 (54°C, 50 μL, Mg2+)
6.	Sample 2.2 (51.9°C, 50 μL, Mg2+)
7.	Sample 2.3 (47.6°C, 50 μL, Mg2+)
8.	Sample 2.4 (44.7°C, 50 μL, Mg2+)
9.	Sample 3.1 (54°C, 75 μL, Mg2+)
10.	Sample 3.2 (51.9°C, 75 μL, Mg2+)
11.	Sample 3.3 (47.6°C, 75 μL, Mg2+)
12.	Sample 3.4 (44.7°C, 75 μL, Mg2+)

Components	Volume for 50 μL reaction
DNA from PCR 1	5 μg
Lambda Exonuclease Reaction Buffer (10X)	5 μL (1X)
Lambda Exonuclease	1 μL (5 units)
Nuclease-free water	up to 50μL

Sample	Pool 1 Elution 1	Pool 1 Elution 2	Pool 2 Elution 1	Pool 2 Elution 2
Upper aqueous phase recovered	2000 µL	430 µL	1150 µL	415 µL
Sodium acetate 3M to add	200 µL	43 µL	115 µL	41.5 µL
Volume after addition of sodium acetate	2200 µL	473 µL	1265 µL	456.5 µL
Ethanol 100% to add	5500 µL	1182.5 µL	3162.5 µL	1141.25 µL
Total volume	7700 µL	1655.5 µL	4427.5 µL	1597.75 µL

	Volume needed (in μL)
Components	Mix 1 (With DMSO)	Mix 2 (Without DMSO)
Phusion HF buffer 5X	50	50
25 mM dNTPs	5	5
Forward primer	10	10
Reverse primer	10	10
DMSO	7.5	0
Template DNA	22.5	22.5
Phusion DNA polymerase	2.5	2.5
Nuclease-free water	142.5	150
Total volume	250	250

Wells	Purification method	Concentration (ng/μL)	260/280 ratio	260/230 ratio
4	PCR product with DMSO	1157.6	1.64	0.85
5	PCR product without DMSO	1143	1.65	1.18
6	PCR clean up kit from QIAGEN with DMSO	7.9	2.26	-0.32
7	PCR clean up kit from QIAGEN without DMSO	15	2.07	-0.62
8	GE Healthcare columns with DMSO	34.5	0.96	0.34
9	GE Healthcare columns without DMSO	45.9	1.16	0.08
10	Gel extraction kit with DMSO	6.1	-6.66	0.01
11	Gel extraction kit without DMSO	-0.5	0.53	0

	Volume needed (in μL)
Components	Mix 1(10ng)	Mix 2(5ng)	Mix 3(1ng)
Phusion Buffer 10X	100	100	100
dNTPs 25 mM	10	10	10
Forward primer (1/10)	20	20	20
Reverse primer (1/10)	20	20	20
Template DNA (1/1000)	45	22.5	5
Phusion DNA polymerase	5	5	5
Nuclease-free water	300	322.5	340
Total volume	500	500	500

Sample	Concentration(ng/µl	260/280 ratio	260/230 ratio
PCR 8 pooled	1093.9	1.63	1.11

	S aureus
Components	SA 1 (1/10)	SA 2 (1/90)
Phusion buffer	10	10
dNTPs	1	1
Forward primer	2	2
Reverse primer	2	2
Template DNA (1/10)	1	0
Template DNA (1/90)	0	1
Phusion DNA polymerase	0.5	0.5
Nulease-free water	33.5	33.5
Total volume	50	50

Sample	Concetration (ng/μL)	260/280 ratio	260/230 ratio
EFr1 sample 1	17.2	1.46	1.29
EFr1 sample 2	26.5	1.49	1.44
EFr1 sample 3	29.8	1.49	1.47
EFr1 sample 4	37.2	1.56	1.5
SAr1 sample 1	69	1.51	1.54
SAr1 sample 2	61.5	1.75	0.28

Components	SAr1	EFr1
Phusion buffer	200	200
dNTPs	20	20
Forward primer	40	40
Reverse primer	40	40
Template DNA (1/3)	20	20
Phusion DNA polymerase	10	10
Nuclease-free water	670	670
Total volume	1000	1000