I. 1x TAE buffer was prepared by diluting a 50x TAE buffer
II. 1.5 g of Agarose were added to 50 ml of 1x TAE buffer (3%) in a conical flask
III. The solution was placed in a microwave and mixed until solution became clear
IV.
3 microliters of Gel Red were added to the solution
V. A well comb was added to the gel cast, the solution was poured into the gel cast and allowed to solidify
VI. After 30 min, 20 drops of 5 ul of loading dye was put on paraffin
paper, after which 10 ul of samples from each
PCRed miniprep was added.
VII. 5 ul of ladder was loaded first, followed by 15 ul of samples in the order: pcaA MP1, pcaA MP2, pcaA MP3, pcaA
negative control, HbcAg MP1, HbcAg
MP2, HbcAg MP3, HbcAg negative control, IS481 MP1, IS481 MP2, IS481
MP3, IS481 negative control, IS481 old MP1, IS481 old MP2, IS481 old MP3, IS481 old negative, ypo MP1, ypo
MP2, ypo MP3, ypo negative control
VIII. The gels
were left to run for 20 minutes and was later viewed under UV light
1. Dilute 100μM FIP, BIP, F3, B3 primers to be 10μM following last year's LAMP optimization.
2. Mix them in one ependorff tube.
3. Pipetted up and down to mixDilution on IS481* MiniPrep2.
1. Added 380 ul of TE buffer to lyophilized quencher to get it to a working stock of 100 uM
2. Took 10 ul of 100 uM and added 10 ul of water to get a stock of 50 uM in 20 ul
3. Took 2 ul of 50 uM solution and added 98 ul of water
to get a concentration of 1uM in 100 ul
Tuesday: 9/17/2019
Serial dilutions for ypo2088 Miniprep 3:
Overnight PCR for ypo2088 post serial dilution samples:
1.Added 20 ul PCR Mastermix to each PCR tube (not enough mastermix was left for the negative control/ just added what was left around 10ul)
2. Added 1 ul
of 10 uM forward primers to each tube.
3. Added 1 ul of 10 uM reverse primers to each tube.
4. Added 2ul of each dilution to each tube. None was added to negative control.
5. Water was added to reach a total volume ot 40 ul
(16 ul for each tube with DNA and 18 ul for negative control)
6. PCR temperatures were set on machine according to BioRad protocol and 35 cycles were done.
7. PCR was left overnight.
Serial dilution for IS481* mp2
Overnight PCR for IS481* mp2 post serial dilution samples:
1. Added 20 ul PCR Mastermix to each PCR tube (not enough mastermix was left for the negative control/ just added what was left around 10ul)
2. Added 1 ul
of 10 uM forward primers to each tube.
3. Added 1 ul of 10 uM reverse primers to each tube.
4. Added 2ul of each dilution to each tube. None was added to negative control.
5. Water was added to reach a total volume ot 40 ul
(16 ul for each tube with DNA and 18 ul for negative control)
6. PCR temperatures were set on machine according to BioRad protocol and 35 cycles were done.
7. PCR was left overnight.
10μl final volume RPA for HbcAg (done with 2 sets of RPA primers), IS481* (done with 2 sets of RPA primers), ypo2088 (done
with first set of primers only) and pcaA (done with first set of primers only)
Protocal
followed when doing HbcAg and IS481* RPAs:
1. Reaction mix in 1.5 mL tube:
a. Primer A (10μM) - 4.8μLr
b. Primer B (10μM) - 4.8μL
c. Rehydration Buffer - 59μL
d. dH2O - 16.4μL
2. Pipetted up and down after addition of
each component in step 1
3. Splitted the reaction mix in two (42.5μL) and added each half to 1 freeze dried reaction. Pipetted up and down to mix.
4. Splitted the reaction into 9 volumes - 8.5μL to 9 separate PCR tubes.
5. Added 1μL of template
from each serial dilution in corresponding tube.
6. Added 0.5μL of 280mM magnesium acetate and mixed well to start the reaction.
7. Incubated at 38°C for 40 min using thermocycler
Protocol followed when doing ypo2088 and pcaA RPAs :
1. Reaction mix was done in 2 different 1.5mL eppendorf tube:
a. Primer A (10μM) - 4.8μL
b. Primer B (10μM) - 4.8μL
c. Rehydration Buffer - 59μL
d. dH2O
- 16.4μL
2. Pipetted up and down after addition of each component in step 1
3. The 8.5reaction mix ( for ypo (for fwd primer 1 and rev 1 primer) was added to the 9 freezed dried reaction (including Negative
control) . Pipette
up and down to mix.
4. Repeat step 3 for pcaA.
5. Added 1μL of template from each serial dilution to both set corresponding tubes.
6. Added 0.5μL of 280mM magnesium acetate and mixed well to start the reaction.
7. Incubated
at 38°C for 40 min using thermocycler
PCR of pcaA post serial dilutions
TE buffer preparation:
1. Added 20 ml of 50x TE buffer to 980 ml of water to make 1x buffer
Agarose Gel Preparation:
a. Prepared a 1x agrose gel by adding
0.5 grams of agrose to 50 ml of TAE buffer.
b. Heating in the solution up in 20 second interval (in the microwave) and stop after making sure that the solution is clear
enough
Sample Loading:
1. 10 μl of
samples from each PCRed diluted sample was added onto a piece of parafilm.
2. 5 μl of purple loading dye was added to each sample drop and mixed by pipetting up and down.
3. 10 ul of 500bp ladder was mixed with 5 ul of green
dye and loaded first followed by 15 ul of samples in the
order: 1, 2, 3, 4, 5, 6, 7, 8, 9
4. The gels were left to run for 20 minutes
Results
Ladder-60 ng/ul dilution- 25ng/ul dilution- 10 ng/ul dilution- 5 ng/ul dilution- 1 ng/ul dilution- 0.1 ng/ul dilution- 0.01 ng/ul dilution- 0.01
ng/ul dilution- negative control
Wednesday: 9/18/2019
Gel Electrophoresis of RPA samples of diluted is481* and HbcAg from day before:
Agarose Gel Preparation:
a. Prepared a 1x agrose gel by adding 0.5 grams of agrose to 50 ml of TAE buffer.
b. Heating in the solution
up in 20 second interval (in the microwave) and stop after making sure that the solution is clear
enough
Sample Loading:
1. 5 μl of samples from each RPA sample was added onto a piece of parafilm.
2. 1
μl of purple loading dye was added to each sample drop and mixed by pipetting up and down.
3. 1 ul of 500bp ladder (molecular ruler) was loaded first (for HbcAg, pre-dyed ladder was used, for ypo, 1 μl of ladder was mixed with 5
μl of purple dye), followed by 6 ul of samples in the order:
a. IS481: ladder, pcr sample 1 - 9 corresponding to the dilutions from high to low concentrations
b. HbcAg: ladder, pcr sample 1 - 9 corresponding to the dilutions
from high to low concentrations
4. The gels were left to run for 20 minutes
Results:
IS481* RPA post serial dilution
Left: 2nd primer set: Ladder-60 ng/ul dilution- 25ng/ul dilution- 10 ng/ul dilution- 5 ng/ul dilution- 1 ng/ul dilution- 0.1 ng/ul dilution- 0.01
ng/ul dilution- 0.01 ng/ul dilution- negative control
Right: 1st primer set: Ladder-60 ng/ul dilution- 25ng/ul dilution- 10 ng/ul dilution- 5 ng/ul dilution- 1 ng/ul dilution- 0.1 ng/ul dilution- 0.01
ng/ul dilution- 0.01 ng/ul dilution- negative control
HBcAg post serial dilutions RPA
Left: 1st primer set: Ladder-60 ng/ul dilution- 25ng/ul dilution- 10 ng/ul dilution- 5 ng/ul dilution- 1 ng/ul dilution- 0.1 ng/ul dilution- 0.01
ng/ul dilution- 0.01 ng/ul dilution- negative control
Right: 2nd primer set: Ladder-60 ng/ul dilution- 25ng/ul dilution- 10 ng/ul dilution- 5 ng/ul dilution- 1 ng/ul dilution- 0.1 ng/ul dilution- 0.01
ng/ul dilution- 0.01 ng/ul dilution- negative control
Thursday: 9/19/2019
Gel Electrophoresis of RPA samples of serial dilutions of ypo2088 from day (17/10/19)
Agarose Gel Preparation:
a. Prepared a 3% agrose gel by adding 1.5 grams of agrose to 50 ml of TAE buffer.
b.
Heating in the solution up in 20-second interval (in the microwave) and stop after making sure that the solution is clear enough. In sum, it took 50seconds to obtain a clear solution.
c. Pour the warm liquid agarose. Place the comb
into the casting tray by placing the sides into the notches. . Wait until the gel polymerizes.
Sample loading
1. 5 μl of samples from each serial dilution of RPA samples of ypo2088 was added onto a piece
of parafilm.
2. 1 μl of purple loading dye was added to each sample drop and mixed by pipetting up and down.
3. Pour the warm liquid agarose. Place the comb into the casting tray by placing the sides into the notches. Wait
until the gel polymerizes.
a. ypo: ladder, rpa serial dilutions sample 1 - 9 corresponding to the dilutions from high to low
concentrations.
4. The gels were left to run for 20 minutes
Result:
Ladder-60 ng/ul dilution- 25ng/ul dilution- 10 ng/ul dilution- 5 ng/ul dilution- 1 ng/ul dilution- 0.1 ng/ul dilution- 0.01 ng/ul dilution- 0.01
ng/ul dilution- negative control
Gel Electrophoresis of RPA samples of diluted is481* and HbcAg from Sept 17:
Agarose Gel Preparation:
a. Prepared a 1x agrose gel by adding 1.5 grams of agrose to 50 ml of TAE buffer.
b. Heating in the solution
up in 20 second interval (in the microwave) and stop after making sure that the solution is clear
enough
Sample Loading:
1. 5 μl of samples from each RPA diluted sample was added onto a piece of parafilm.
2.
1 μl of purple loading dye was added to each sample drop and mixed by pipetting up and down.
3. 1 ul of 100bp ladder (molecular ruler) was loaded first (1 μl of ladder was mixed with 5 μl of purple dye), followed by 6 ul of samples
in the order:
a. IS481: ladder, rpa with primer set 1 sample 1 - 9 corresponding to the dilutions from high to low concentrations, empty space, ladder, rpa with primer set 2 sample 1-9 correspondinG to dilutiosn from high to low
conc.
b. HbcAg: ladder, rpa sample 1 - 9 corresponding to the dilutions from high to low concentrations, empty space, ladder, rpa with primer set 2 sample 1-9 correspondinG to dilutiosn from high to low conc.
4. The gels were
left to run for 20 minutes
Results:
IS481* post serial dilution RPA
Ladder-60 ng/ul dilution- 25ng/ul dilution- 10 ng/ul dilution- 5 ng/ul dilution- 1 ng/ul dilution- 0.1 ng/ul dilution- 0.01 ng/ul dilution- 0.01
ng/ul dilution- negative control
HBcAg post serial dilution RPA
Left: 1st primer set: Ladder-60 ng/ul dilution- 25ng/ul dilution- 10 ng/ul dilution- 5 ng/ul dilution- 1 ng/ul dilution- 0.1 ng/ul dilution- 0.01
ng/ul dilution- 0.01 ng/ul dilution- negative control
Right: 2nd primer set: Ladder-60 ng/ul dilution- 25ng/ul dilution- 10 ng/ul dilution- 5 ng/ul dilution- 1 ng/ul dilution- 0.1 ng/ul dilution- 0.01
ng/ul dilution- 0.01 ng/ul dilution- negative control
Serial dilutions for HbcAg mp2:
10μl final volume RPA for HbcAg (done with 2 sets of RPA primers), IS481* (done with 2 sets of RPA primers), pcaA (1st set of
primers)
Protocal followed when doing HbcAg and IS481* RPAs:
1. Reaction mix in 1.5 mL tube:
a. Primer A (10μM) - 4.8μL
b. Primer B (10μM) - 4.8μL
c. Rehydration Buffer - 59μL
d. dH2O - 16.4μL
2. Pipetted up and down after addition
of each component in step 1
3. Splitted the reaction mix in two (42.5μL) and added each half to 1 freeze dried reaction. Pipetted up and down to mix.
4. Splitted the reaction into 9 volumes - 8.5μL to 9 separate PCR tubes.
5.
Added 1μL of template from each serial dilution in corresponding tube.
6. Added 0.5μL of 280mM magnesium acetate and mixed well to start the reaction.
7. Incubated at 38°C for 40 min using thermocycler
Gel Electrophoresis of RPA samples of diluted is481* and HbcAg from above-said RPA run:
Agarose Gel Preparation:
a. Prepared a 1x agrose gel by adding 1.5 grams of agrose to 50 ml of TAE buffer.
b.
Heating in the solution up in 20 second interval (in the microwave) and stop after making sure that the solution is clear
enough
Sample Loading:
1. 5 μl of samples from each RPA diluted sample was added onto
a piece of parafilm.
2. 1 μl of purple loading dye was added to each sample drop and mixed by pipetting up and down.
3. 1 ul of 100bp ladder (molecular ruler) was loaded first (1 μl of ladder was mixed with 5 μl of purple dye),
followed by 6 ul of samples in the order: IS481:
a. ladder, rpa with primer set 1 sample 1 - 9 corresponding to the dilutions from high to low
concentrations, empty space, ladder, rpa with primer set 2 sample 1-9 correspondin
to dilutiosn from high to low conc.
b. HbcAg: ladder, rpa sample 1 - 9 corresponding to the dilutions from high to low concentrations, empty space, ladder, rpa with primer set 2 sample 1-9 correspondinG to dilutiosn from high to
low conc.
4. The gels were left to run for 20 minutes
Friday: 9/20/2019
Gel Electrophoresis of RPA samples of diluted is481* fom Sept 19:
Agarose Gel Preparation:
a. Prepared a 1x agrose gel by adding 1.5 grams of agrose to 50 ml of TAE buffer.
b. Heating in the solution up in 20
second interval (in the microwave) and stop after making sure that the solution is clear
enough
Sample Loading:
1. 5 μl of samples from each RPA diluted sample was added onto a piece of parafilm.
2. 1 μl
of purple loading dye was added to each sample drop and mixed by pipetting up and down.
3. 1 ul of 100bp ladder (molecular ruler) was loaded first (1 μl of ladder was mixed with 5 μl of purple dye),
followed by 6 ul of samples
in the order:
a.IS481: ladder, rpa with primer set 1 sample 1 - 9 corresponding to the dilutions from high to low concentrations, empty space, ladder, rpa with primer set 2 sample 1-9 correspondinG to dilutiosn from high to low
conc.
b. HbcAg: ladder, rpa sample 1 - 9 corresponding to the dilutions from high to low concentrations, empty space, ladder, rpa with primer set 2 sample 1-9 correspondinG to dilutiosn from high to low conc.
4. The gels were
left to run for 20 minutes
RPA for post dilution IS481* (both primer 1 and 2 sets)
Protocal followed when doing IS481* RPAs:
1. Reaction mix in 1.5 mL tube:
a. Primer A (10μM) - 4.8μL
b. Primer B (10μM) - 4.8μL
c. Rehydration Buffer
- 59μL
d. dH2O - 16.4μL
2. Pipetted up and down after addition of each component in step 1
3. Splitted the reaction mix in two (42.5μL) and added each half to 1 freeze dried reaction. Pipetted up and down to mix.
4. Splitted
the reaction into 9 volumes - 8.5μL to 9 separate PCR tubes.
5. Added 1μL of template from each serial dilution in corresponding tube.
6. Added 0.5μL of 280mM magnesium acetate and mixed well to start the reaction.
7. Incubated
at 38°C for 40 min using thermocycler
Gel Electrophoresis of RPA samples of diluted is481* from above-said RPA run:
Agarose Gel Preparation:
a. Prepared a 1x agrose gel by adding 1.5 grams of agrose to 50 ml of TAE buffer.
b. Heating in the solution
up in 20 second interval (in the microwave) and stop after making sure that the solution is clear
enough
Sample Loading:
1. 5 μl of samples from each RPA diluted sample was added onto a piece of parafilm.
2. 1 μl of purple
loading dye was added to each sample drop and mixed by pipetting up and down.
3. 1 ul of 100bp ladder (molecular ruler) was loaded first (1 μl of ladder was mixed with 5 μl of purple dye), followed by 6 ul of samples in the order:
a.
IS481: ladder, rpa with primer set 1 sample 1 - 9 corresponding to the dilutions from high to low concentrations, empty space, ladder, rpa with primer set 2 sample 1-9 correspondinG to dilutiosn from high to low conc.
b. HbcAg:
ladder, rpa sample 1 - 9 corresponding to the dilutions from high to low concentrations, empty space, ladder, rpa with primer set 2 sample 1-9 correspondinG to dilutiosn from high to low conc.
4. The gels were left to run for 20
minutes
Saturday: 9/21/2019
Agarose Gel Preparation:
a. Prepared a 1x agrose gel by adding 1.5 grams of agrose to 50 ml of TAE buffer.
b. Heating in the solution up in 20 second interval (in the microwave) and stop after making sure that the
solution is clear
enough
Sample Loading: (PCR post serial dilutions for ypo2088 Sept 17th)
1. 5 μl of samples from each PCRed diluted sample was added onto a piece of parafilm.
2. 1 μl of purple loading dye was added to
each sample drop and mixed by pipetting up and down.
3. 1 ul of 500bp ladder was mixed with 5 ul of dye and loaded first followed by 15 ul of samples in the order: 1, 2, 3, 4, 5, 6, 7, 8, 9
4. The gels were left to run for 20
minutes
RPA ypo2088 1st primer set:
Reaction mix was done in 2 different 1.5mL eppendorf tube:
I. Primer A (10μM) - 4.8μL
II. Primer B (10μM) - 4.8μL
III. Rehydration Buffer - 59μL
IV. dH2O - 16.4μL
a. Pipetted
up and down after addition of each component in step 1
b. The 8.5reaction mix ( for ypo (for fwd primer 1 and rev 1 primer) was added to the 9 freezed dried reaction (including Negative control) . Pipette up and down to mix.
c.
Repeat step 3 for pcaA.
d. Added 1μL of template from each serial dilution to both set corresponding tubes.
e. Added 0.5μL of 280mM magnesium acetate and mixed well to start the reaction.
f. Incubated at 38°C for 40 min using
thermocycler
10μl final volume RPA for pcaA (2nd set of primers)
Protocal followed when doing HbcAg and IS481* RPAs:
a. Reaction mix in 1.5 mL tube:
I. Primer A (10μM) - 4.8μL
II. Primer B (10μM) - 4.8μL
III. Rehydration
Buffer - 59μL
IV. dH2O - 16.4μL
b. Pipetted up and down after addition of each component in step 1
c. Splitted the reaction mix in two (42.5μL) and added each half to 1 freeze dried reaction. Pipetted up and down to mix.
d.
Splitted the reaction into 9 volumes - 8.5μL to 9 separate PCR tubes.
e. Added 1μL of template from each serial dilution in corresponding tube.
f. Added 0.5μL of 280mM magnesium acetate and mixed well to start the reaction.
g.
Incubated at 38°C for 40 min using thermocycler
Gel Electrophoresis of RPA samples of diluted HbcAg from above-said RPA run:
Agarose Gel Preparation:
I. Prepared a 1x agrose gel by adding 1.5 grams of agrose to 50 ml of TAE buffer.
Heating in the solution up
in 20 second interval (in the microwave) and stop after making sure that the solution is
clear enough
Sample Loading:
a. 5 μl of samples from each RPA diluted sample was added onto a piece of parafilm.
b. 1 μl of purple loading dye was added to each sample drop and mixed by pipetting up and down.
c. 1 ul
of 100bp ladder (molecular ruler) was loaded first (1 μl of ladder was mixed with 5 μl of purple
dye), followed by 6 ul of samples in the order:
d. IS481: ladder, rpa with primer set 1 sample 1 - 9 corresponding to the dilutions
from high to low concentrations, empty space, ladder, rpa with primer set 2 sample 1-9 correspondinG to dilutiosn from high to low conc.
e. HbcAg: ladder, rpa sample 1 - 9 corresponding to the dilutions from high to low concentrations,
empty space, ladder, rpa with primer set 2 sample 1-9 correspondinG to dilutiosn from high to low conc.
f. The gels were left to run for 20 minutes
Sunday: 9/22/2019
CRISPR of RPA samples of diluted is481* and HbcAg from above-said RPA run:
CRISPR reaction following NEB protocol:
1. Assemble the reaction at room temperature in the following order*:
a. 20μl Nuclease-free water
b.
3μl NEBuffer 2.1 Reaction Buffer (10x)
c. 3μl 300nM gRNA
d. 1μl 1 μM EnGen Lba Cas12a (Cpf1)
2. Pre-incubate for 10 minutes at 250C.
3. Add 3 μl of substrate DNA (30 μl final volume).
4. Vortex and pulse-spin in a microfuge.
5.
Incubate at 37°C for 10 minutes.
6. Add 1.5 μl of 1uM FQ quencher
7. Incubate for 60 minutes at 37°C. Check the fluorescence every 10 minutes using fluorescent microscope.
Serial Dilution of ypo2088, MP1:
Resuspension of RPA rev and fwd 2 for ypo2088 according to the order sheet.
10μl final volume RPA for ypo2088 (2nd set of primers)
a. Reaction mix in 1.5 mL tube:
I. Primer A (10μM) - 4.8μL
II. Primer B (10μM) - 4.8μL
III. Rehydration Buffer - 59μL
IV. dH2O - 16.4μL
b. Pipetted
up and down after addition of each component in step 1
c. Splitted the reaction mix in two (42.5μL) and added each half to 1 freeze dried reaction. Pipetted up and down to mix.
d. Splitted the reaction into 9 volumes - 8.5μL
to 9 separate PCR tubes.
e. Added 1μL of template from each serial dilution in corresponding tube.
f. Added 0.5μL of 280mM magnesium acetate and mixed well to start the reaction.
g. Incubated at 38°C for 30 min using thermocycler
Monday: 9/23/2019
CRISPR cas 12a on optimized RPA samples out of the serial dilutions
10μl final volume RPA for ypo2088 (1st & 2nd set of primers)
Reaction mix was done in 2 different 1.5mL eppendorf tube:
1. Primer A (10μM) -
4.8μL
2. Primer B (10μM) - 4.8μL
3. Rehydration Buffer - 59μL
4. dH2O - 16.4μL
I. Pipetted up and down after addition of each component in step 1
The 8.5reaction mix ( for ypo (for fwd primer 1 and rev 1 primer) was added to the
9 freezed dried reaction
(including Negative control) . Pipette up and down to mix.
II.
III. Repeat step 3 for ypo with primers set 2 ( fwd primer 2 and rev primer 2)
IV. Added 1μL of template from each serial dilution to both
set corresponding tubes.
V. Added 0.5μL of 280mM magnesium acetate and mixed well to start the reaction.
VI. Incubated at 38°C for 40 min using thermocycler
Cas12 Cleavage with ypo 2088 gene(serial dilutions) :
1. Making 125nM gRNA: 0.2 μl of 1000 μM gRNA and 1.58 ml of water were mixed
2. Making 100nM CRISPR LbCas12a: 1 μl of 100 μM Cas12 and 999 μl of water
3.
1 μl of each solution were used to make 2 μl 62.5nM gRNA : 50nM Cas12a solution and added to approximately 18 μl of Cra DNA solution.
4. The same solution was made with 1 μl each of undiluted Cas12a and gRNA solutions (gRNA:
1000 μM; CAS12a: 100 μM).
5. The solutions were left to set for 1.5 hours in 37°C incubator.
LAMP on ypo
Preparation of LAMP Primer Mix ypo2088
1. Dilute 100μM FIP, BIP, F3, B3 primers to be 10μM following last year's LAMP optimization.
2. Mix them in one ependorff tube.
3. Pipetted
up and down to mix
Preparation of LAMP Primer Mix stock (total volume 100μl) for HBcAg and IS481
1. Add 4μl of FIP (100μM), 4μl of BIP (100μM), 1μl of F3 (100μM),, 1μl of B3 (100μM), and 45μl of dH2O in the
ependorff tube.
2. Pipetted up and down to mix.
3. Vortex the primer mix.
LAMP reaction for HBcAg and IS481* post serial dilution samples
1. Add 15μl of isothermal master mix to each tube (total 9 tubes
for every gene).
2. Add 5μl of the primer mix to each tube.
3. Add 5μl of the dilution sample to each tube (8 dilution samples + 1 negative control each gene).
4. Incubate at 65C for 20 minutes.
● Gel visualization
a. 5 μl of samples from each RPA diluted sample+ CRISPR cas 12a was added onto a piece of parafilm.
b. 1 μl of purple loading dye was added to each sample drop and mixed by pipetting up and down.
c. 1 ul of 100bp ladder (molecular
ruler) was loaded first (1 μl of ladder was mixed with 5 μl of purple dye), followed by 6 ul of samples in the order: ypo gene: ladder, empty space, lamp primers 1-8, negative control and rpa with primer set 1 sample 1 - 5 corresponding
to the dilutions from high to low concentrations.
RPA on IS4
RPA and CRISPR on HbcAg MP1 samples (1st and 2nd set of primers):
RPA:
1. reaction mix in 2 different 1.5 eppendorf tube:
a. Primer A (10μM) - 2.4μL
b. Primer B (10μM) - 2.4μL
c.
Rehydration Buffer - 29.5μL
d. dH2O - 8.5μL
e. sample - 5μL
2. Pipetted up and down after addition of each component in step 1
3. Added 2.5μL of 280mM magnesium acetate and mixed well to start the reaction.
4. Incubated
at 38°C for 30 min using thermocycler
CRISPR following NEB protocal:
1. Assemble the reaction at room temperature in the following order*:
a. 20μl Nuclease-free water
b. 3μl NEBuffer 2.1 Reaction Buffer
(10x)
c. 3μl 300nM gRNA
d. 1μl 1 μM EnGen Lba Cas12a (Cpf1)
2. Pre-incubate for 10 minutes at 250C.
3. Added 3 μl of substrate DNA (30 μl final volume).
4. Vortex and pulse-spin in a microfuge.
5. Incubate at
37°C for 10 minutes.
6. Added 1.5 μl of 1uM FQ quencher
7. Incubated for 60 minutes at 37°C. Check the fluorescence every 10 minutes using fluorescent microscope.
Results: No fluorescence can be seen.
Monday: 9/24/2019
50μl final volume RPA for pcaA (1st set of primers)
Protocal followed when doing HbcAg and IS481* RPAs:
a. Reaction mix in 1.5 mL tube:
I. Primer A (10μM) - 21.6μL
II. Primer B (10μM) - 21.6μL
III. Rehydration Buffer - 265.5 μL
IV. dH2O - 118.8 μL
b. Pipetted up
and down after addition of each component in step 1
c. Splitted the reaction mix in nine (47.5μL) and added each to 1 freeze dried reaction. Pipetted up and down to mix.
d. Added 1ul of DNA (with buffer) into each tube
e.
Added 2.5μL of 280mM magnesium acetate and mixed well to start the reaction.
f. Incubated at 39°C for 20 min using thermocycler
10μl final volume RPA for HbcAg (done with 2 sets of RPA primers) and pcaA (2nd set)
Protocal followed when doing HbcAg and IS481* RPAs:
1. Reaction mix in 1.5 mL tube: (2 tubes, 1 for each primer pair)
a. Primer A (10μM) - 4.8μL
b. Primer B (10μM) - 4.8μL
c. Rehydration Buffer - 59μL
d. dH2O - 16.4μL
2.
Pipetted up and down after addition of each component in step 1
3. Splitted the reaction mix in two (42.5μL) and added each half to 1 freeze dried reaction. Pipetted up and down to mix.
4. Splitted the reaction into 9
volumes - 8.5μL to 9 separate PCR tubes.
5. Added 1μL of template from each serial dilution in corresponding tube.
6. Added 0.5μL of 280mM magnesium acetate and mixed well to start the reaction.
7. Incubated at 38°C
for 30 min using thermocycler
Gel Electrophoresis of RPA samples of diluted HbcAg from above-said RPA run:
Agarose Gel Preparation:
I. Prepared a 1x agrose gel by adding 1.5 grams of agrose to 50 ml of TAE buffer.
II. Heating in the solution up in 20 second interval (in the microwave) and stop after making sure
that the solution is
clear enough
Sample Loading:
a. 5 μl of samples from each RPA diluted sample was added onto a piece of parafilm.
b. 1 μl of purple loading dye was added to each sample drop
and mixed by pipetting up and down.
c. 5 ul of 100bp ladder (molecular ruler) was loaded first (5 μl of ladder was mixed with 1 μl of purple
dye), followed by 6 ul of samples in the order:
d. HbcAg: space, ladder,
rpa sample 1 - 9 corresponding to the dilutions from high to low concentrations, empty space, ladder, rpa with primer set 2 sample 1-9 correspondinG to dilutiosn from high to low conc.
e. The gels were left to run for 20
minutes
Serial Dilution for pcaA (Using Miniprep 1-A)
IS4
Wednesday : 9/25/2019
PCR of HbcAg Serial Dilutionsa and 2 cra samples (extracted from IS481* agar plate)
(following Invitrogen Taq DNA polymerase protocal)
In 1 eppendorf tube added:
○ 2.5 μl 10X PCR Buffer
○ 0.75 μl 50mM MgCl2
○ 2 μl 2mM dNTP Mix
○ 1.25 μl 10 μM forward primer
○ 1.25 μl 10 μM reverse primer
○
0.1 μl Taq DNA Polymerase
○ for Cra:
■ added 18.15μl distilled water
■ scraped colony off of IS481* agar plate to extract Cra gene (Cra is endogenous to E.coli)
○ for HbcAg:
■ added 16.15μl distilled wtaer
PCR
was left overnight.
LAMP for YPO 2088 . repeated
Thursday : 9/26/2019
50μl final volume RPA for is481 (1st set of primers)
Protocal followed when doing HbcAg and IS481* RPAs:
I. Reaction mix in 1.5 mL tube:
1. Primer A (10μM) - 21.6μL
2. Primer B (10μM) - 21.6μL
3. Rehydration Buffer - 265.5 μL
4. dH2O - 118.8 μL
II. Pipetted up and down after addition of each component in step 1
III.
Splitted the reaction mix in nine (47.5μL) and added each to 1 freeze dried reaction. Pipetted up and down to mix.
IV. Added 5ul of DNA (with buffer) into each tube
V. Added 2.5μL of 280mM magnesium acetate and mixed
well to start the reaction.
VI. Incubated at 38°C for 30 min using thermocycler
CRISPR of RPA samples of cra after RPA:
CRISPR reaction following NEB protocol:
a. Assemble the reaction at room temperature in the following order*:
I. 20μl Nuclease-free water
II. 3μl NEBuffer 2.1
Reaction Buffer (10x)
III. 3μl 300nM gRNA
IV. 1μl 1 μM EnGen Lba Cas12a (Cpf1)
b. Pre-incubate for 10 minutes at 250C.
c. Add 3 μl of substrate DNA (30 μl final volume).
d. Vortex and pulse-spin in a microfuge.
e.
Incubate at 37°C for 10 minutes.
f. Add 1.5 μl of 1uM FQ quencher
g. Incubate for 60 minutes at 37°C. Check the fluorescence every 10 minutes using fluorescent microscope.
HbcAg RPA (2nd primer set)
a. Reaction mix in 1.5 mL tube:
1. Primer A (10μM) - 4.8μL
2. Primer B (10μM) - 4.8μL
3. Rehydration Buffer - 59μL
4. dH2O - 16.4μL
I. Pipetted up and down after addition
of each component in step 1
Splitted the reaction mix in two (42.5μL) and added each half to 1 freeze dried reaction. Pipetted up and down to
mix.
II.
III. Splitted the reaction into 9 volumes - 8.5μL to 9 separate
PCR tubes.
IV. Added 1μL of template from each serial dilution in corresponding tube.
V. Added 0.5μL of 280mM magnesium acetate and mixed well to start the reaction.
VI. Incubated at 38°C for 30 min using thermocycler
Gel Electrophoresis of HbcAg RPA (2nd primer set) and PCR from previous day
Agarose Gel Preparation:
1.
a. RPA gel: Prepared a 1x agrose gel by adding 1.5 grams of agrose to 50 ml of TAE buffer.
b.
PCR gel: Prepared a 1x agrose gel by adding 0.5 grams of agrose to 50 ml of TAE buffer.
2. Heating in the solution up in 20 second interval (in the microwave) and stop after making sure that the solution is clear enough
Sample Loading:
a.
5 μl of samples from each RPA diluted sample was added onto a piece of parafilm.
b. 1 μl of purple loading dye was added to each sample drop and mixed by pipetting up and down.
c. 5 ul of a) 100bp ladder (molecular
ruler) for RPA gel b) 500bp ladder for PCR gel were loaded first (5 μl of ladder was mixed with 1 μl of purple dye), followed by 6 ul of samples in the order from high to low conc.
d. The gels were left to run for 20 minutes
HbcAg Serial Dilution (MP3)
IS4
2nd HbcAg RPA (2nd primer set)
I. Reaction mix in 1.5 mL tube:
a. Primer A (10μM) - 4.8μL
b. Primer B (10μM) - 4.8μL
c. Rehydration Buffer - 59μL
d. dH2O - 16.4μL
1. Pipetted up and down after
addition of each component in step 1
2.Splitted the reaction mix in two (42.5μL) and added each half to 1 freeze dried reaction. Pipetted up and down to mix.
3. Splitted the reaction into 9 volumes - 8.5μL to 9 separate
PCR tubes.
4. Added 1μL of template from each serial dilution in corresponding tube.
5. Added 0.5μL of 280mM magnesium acetate and mixed well to start the reaction.
6. Incubated at 38°C for 30 min using thermocycler
2nd HbcAg and Cra (1st primer set) PCR using 2x mastermix
I. Labelled each eppendorf tube with name of gene and the concentration
II. Added 20 ul PCR Mastermix to each tube
III. Added 1 ul of 10 uM forward
primers to each tube.
IV. Added 1 ul of 10 uM reverse primers to each tube.
V. Added 2ul of each dilution to each tube. None was added to negative control.
1. For cra samples, colonies are scraped from IS481* plate
and put directly into the master mix
VI. Water was added to reach a total volume ot 40 ul (16 ul for each tube with DNA and 18 ul for negative control and Cra sample tubes)
VII. PCR temperatures were set on machine
according to BioRad protocol and 35 cycles were done.
VIII. PCR was left overnight.
Gel Electrophoresis of 2nd HbcAg RPA (2nd primer set)
Agarose Gel Preparation:
a. RPA gel: Prepared a 1x agrose gel by adding 1.5 grams of agrose to 50 ml of TAE buffer.
b. Heating in
the solution up in 20 second interval (in the microwave) and stop after making sure that the solution is clear enough
Sample Loading:
a. 5 μl of samples from each RPA diluted sample was added onto a piece
of parafilm.
b. 1 μl of purple loading dye was added to each sample drop and mixed by pipetting up and down.
c. 5 ul of a) 100bp ladder (molecular ruler) for RPA gel b) 500bp ladder for PCR gel were loaded first (5 μl
of ladder was mixed with 1 μl of purple dye), followed by 6 ul of samples in the order from high to low conc.
d. The gels were left to run for 20 minutes
Friday : 9/27/2019
HbcAg RPA (2nd primer set)
I. Reaction mix in 1.5 mL tube:
a. Primer A (10μM) - 4.8μL
b. Primer B (10μM) - 4.8μL
c. Rehydration Buffer - 59μL
d. dH2O - 16.4μL
1. Pipetted up and down after
addition of each component in step 1
2. Splitted the reaction mix in two (42.5μL) and added each half to 1 freeze dried reaction. Pipetted up and down to mix.
3. Splitted the reaction into 9 volumes - 8.5μL to 9 separate
PCR tubes.
4. Added 1μL of template from each serial dilution in corresponding tube.
5. Added 0.5μL of 280mM magnesium acetate and mixed well to start the reaction.
6. Incubated at 39°C for 30 min using thermocycler
Gel Electrophoresis of HbcAg RPA (2nd primer set) from today and PCR from previous day
Agarose Gel Preparation:
1.
a. RPA gel: Prepared a 1x agrose gel by adding 1.5 grams of agrose to
50 ml of TAE buffer.
b. PCR gel: Prepared a 1x agrose gel by adding 0.5 grams of agrose to 50 ml of TAE buffer.
2.Heating in the solution up in 20 second interval (in the microwave) and stop after making sure that the
solution is clear enough
Sample Loading:
a. 5 μl of samples from each RPA diluted sample was added onto a piece of parafilm.
b. 1 μl of purple loading dye was added to each sample drop and mixed by
pipetting up and down.
c. 5 ul of a) 100bp ladder (molecular ruler) for RPA gel b) 500bp ladder for PCR gel were loaded first (5 μlof ladder was mixed with 1 μl of purple dye), followed by 6 ul of samples in the order from
high to low conc.
d. The gels were left to run for 20 minutes
Saturday : 9/28/2019
NEB Protocol for RPA on pcaA (for the engineers- total volume is 50 ul) done at 10 min and 20 min
1. Prepare reaction mix in a 1.5 ml tube:
a. Primer A (10 ul): 2.4 ul
b. Primer B (10 ul): 2.4 ul
c.
Rehydration buffer: 29.5 ul
d. Nuclease free water: 11.2 ul
e. Template: 2 ul
2. Vortex
3. Add mixture to a TwistAmp Basic Reaction
4. Add 2.5 ul of 280mM of Magnesium Acetate
5. Incubate at 39 C for 10/20
minutes
NEB CRISPR + FQ reporter on PCR-amplified Cra (modified)
CRISPR reaction following NEB protocol:
1. Assemble the reaction at room temperature in the following order*:
a. 5μl Nuclease-free water
b. 3μl
NEBuffer 2.1 Reaction Buffer (10x)
c. 3μl 300nM gRNA
d. 1μl 1 μM EnGen Lba Cas12a (Cpf1)
e. 16.5μl 1μM FQ reporter
2.
a. for tube 1, Pre-incubated for 10 minutes at 250C.
b. for tube 2, pre-incubated for 10
minutes at 370C
3. Add 3 μl of substrate DNA (30 μl final volume).
4. Incubate at 37°C for 10 minutes.
5. Incubate for 60 minutes at 37°C. Check the fluorescence every 10 minutes using fluorescent microscope.
Gel Electrophoresis of CRISPR'ed PCR-amplified Cra
Agarose Gel Preparation:
1.
a. PCR gel: Prepared a 1x agrose gel by adding 0.5 grams of agrose to 50 ml of TAE buffer.
2. Heating in the solution up
in 20 second interval (in the microwave) and stop after making sure that the solution is clear enough
Sample Loading:
a. 5 μl of samples from each RPA diluted sample was added onto a piece of parafilm.
b.
1 μl of purple loading dye was added to each sample drop and mixed by pipetting up and down.
c. 5 ul of 500bp ladder for PCR gel were loaded first (5 μl of ladder was mixed with 1 μl of purple dye), followed by 6 ul of samples
in the order:
PCR'ed sample, CRISPR'ed sample with binding incubated at 370C, CRISPR'ed sample with
binding incubated at 250C, negative control
d. The gel was left to run for 20 minutes
Sunday : 9/29/2019
15μl RPA with a)4μl b) 10μl sample DNA (done with HbcAg and IS481)
I. Reaction mix in 1.5 mL tube:
a. Primer A (10μM) - 9.6μL
b. Primer B (10μM) - 9.6μL
c. Rehydration Buffer - 118μL
d. dH2O - 32.8μL
1.
Pipetted up and down after addition of each component in step 1
2. Splitted the reaction mix in four (42.5μL) and added each half to 1 freeze dried reaction. Pipetted up and down to mix.
3. Splitted the reaction into
9 volumes - 15μL to 9 separate PCR tubes.
4. Added:
a. 4μL of template from each serial dilution in corresponding tube.
b. 10μL of template from each serial dilution in corresponding tube
5. Added:
a. 1μL of
280mM magnesium acetate and mixed well to start the reaction.
b. 1.25μL of 280mM magnesium acetate and mixed well to start the reaction.
6. Incubated at 39°C for 30 min using thermocycler
Gel Electrophoresis of HbcAg and IS481 RPA from today and PCR-amplified Cra
Agarose Gel Preparation:
1.
a. RPA gel: Prepared a 3x agrose gel by adding 1.5 grams of agrose to 50 ml of TAE buffer.
b.
PCR gel: Prepared a 1x agrose gel by adding 0.5 grams of agrose to 50 ml of TAE buffer.
2. Heating in the solution up in 20 second interval (in the microwave) and stop after making sure that the solution is clear enough
Sample Loading:
a.
5 μl of samples from each RPA diluted sample was added onto a piece of parafilm.
b. 1 μl of purple loading dye was added to each sample drop and mixed by pipetting up and down.
c. 5 ul of 100bp ladder (molecular ruler)
for RPA gel were loaded first (5 μl of ladder was mixed with 1 μl of purple dye), followed by 6 ul of samples in the order from high to low conc (first samples with 4μl DNA added, then samples with 10 μl added)
d. The gels
were left to run for 20 minutes
Monday : 9/30/2019
Test SYBR Green
Dilute SYBR Green 10,000X to 1,000X using TE buffer
Add SYBR Greeen to yesterday's IS481 RPA samples (both 4μL and 10μL DNA).
1. Adjust the last year's SYBR Green protocol
(1μL of SYBR Green to 25μL sample) for the total sample volume of 14μL and 20μL.
a. Add 0.56μL of SYBR Green to RPA sample with 4μL of DNA
b. Add 0.80μL of SYBR Green to RPA sample with 10μL of DNA
2. Test the fluorescence
under UV light
Dilute SYBR Green to 50X and 100X:
1. 50X SYBR Green: Add 5μL of 1000X SYBR Green to 95μL of TE buffer
2. 100X SYBR Green: Add 10μL of 1000X SYBR Green to 90μL of TE buffer
Test 50X, 100X, and 32X(?) on yesterday's three HbcAg RPA samples (both 4μL and 10μL DNA) that show band in the gel electrophoresis.
1. Total volume of 14μL:
a. 32X:
b. 50X:
c. 100X:
2. Total volume of 20μL:
a.
32X:
b. 50X:
c. 100X:
HbcAg Innoculations:
1. 10mL LB broth was added to 15 15mL culture tubes
2. A plastic inoculation loop was used to select 4 colonies from HbcAg plate and was swirled in the corresponding broth to dislodge
the colony
3. The tubes were loosely capped and incubated on a shaker at 220rpm and 37°C (48 hours)
