Difference between revisions of "Team:Pasteur Paris/test/Notebook"
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<div id="AU3" class="ent" > | <div id="AU3" class="ent" > | ||
| − | + | <p class="p1N"> August Week 3 : 08/19 to | |
| − | + | 08/21 <br> | |
| − | + | <br> | |
| − | + | </p> | |
| − | + | <p class="date">08.19.19</p> | |
| − | + | <p class="p3N">PCR 7 - Generation of new materials (10 ng, 5 ng, 1 | |
| − | + | ng)</p> | |
| − | + | <p class="p2N">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.</p> | |
| − | + | <br> | |
| − | + | <table style="border-color: transparent" class="notTABLE"> | |
| − | + | <tbody> | |
| − | + | <tr class="notTR"> | |
| − | + | <td><br> | |
| − | + | </td> | |
| − | + | <td colspan="3" class="notTD"> Volume needed (in μL)</td> | |
| − | + | </tr> | |
| − | + | <tr class="NotTR"> | |
| − | + | <td class="notTD">Components</td> | |
| − | + | <td class="notTD">Mix 1(10ng)</td> | |
| − | + | <td class="notTD">Mix 2(5ng)</td> | |
| − | + | <td class="not TD">Mix 3(1ng)</td> | |
| − | + | </tr> | |
| − | + | <tr class="notTR"> | |
| − | + | <td class="notTD">Phusion Buffer 10X</td> | |
| − | + | <td class="notTD">100</td> | |
| − | + | <td class="notTD">100</td> | |
| − | + | <td class="notTD">100</td> | |
| − | + | </tr> | |
| − | + | <tr class="notTR"> | |
| − | + | <td class="notTD">dNTPs 25 mM</td> | |
| − | + | <td class="notTD">10</td> | |
| − | + | <td class="notTD">10</td> | |
| − | + | <td class="notTD">10</td> | |
| − | + | </tr> | |
| − | + | <tr class="notTR"> | |
| − | + | <td class="notTD">Forward primer (1/10)</td> | |
| − | + | <td class="notTD">20</td> | |
| − | + | <td class="notTD">20</td> | |
| − | + | <td class="notTD">20</td> | |
| − | + | </tr> | |
| − | + | <tr class="notTR"> | |
| − | + | <td class="notTD"> Reverse primer (1/10)</td> | |
| − | + | <td class="notTD">20</td> | |
| − | + | <td class="notTD">20</td> | |
| − | + | <td class="notTD">20</td> | |
| − | + | </tr> | |
| − | + | <tr class="notTR"> | |
| − | + | <td class="notTD"> Template DNA (1/1000)</td> | |
| − | + | <td class="notTD">45</td> | |
| − | + | <td class="notTD">22.5</td> | |
| − | + | <td class="notTD">5</td> | |
| − | + | </tr> | |
| − | + | <tr class="notTR"> | |
| − | + | <td class="notTD">Phusion DNA polymerase</td> | |
| − | + | <td class="notTD">5</td> | |
| − | + | <td class="notTD">5</td> | |
| − | + | <td class="notTD">5</td> | |
| − | + | </tr> | |
| − | + | <tr class="notTR"> | |
| − | + | <td class="notTD">Nuclease-free water</td> | |
| − | + | <td class="notTD">300</td> | |
| − | + | <td class="notTD">322.5</td> | |
| − | + | <td class="notTD">340</td> | |
| − | + | </tr> | |
| − | + | <tr class="notTR"> | |
| − | + | <td class="notTD">Total volume</td> | |
| − | + | <td class="notTD">500</td> | |
| − | + | <td class="notTD">500</td> | |
| − | + | <td class="notTD">500</td> | |
| − | + | </tr> | |
| − | + | </tbody> | |
| − | + | </table> | |
| − | + | <p class="p2N">Samples were also 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">Sample pooled (10ng)</td> | |
| − | + | <td class="notTD">990.6</td> | |
| − | + | <td class="notTD">1.62</td> | |
| − | + | <td class="notTD">1.05</td> | |
| − | + | </tr> | |
| − | + | <tr class="notTR"> | |
| − | + | <td class="notTD">Sample pooled (5ng)</td> | |
| − | + | <td class="notTD">1028.5)</td> | |
| − | + | <td class="notTD">1.61</td> | |
| − | + | <td class="notTD">1.05</td> | |
| − | + | </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> | ||
</div> | </div> | ||
Latest revision as of 23:17, 21 October 2019
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malesuada ut sodales non, blandit non ligula. Donec venenatis
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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.
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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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.