WEEK 9
TUESDAY : 10/01/2019
15μl RPA with 10μl sample DNA (done with pcaA)
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 20 min using thermocycler
WEDNESDAY : 10/02/2019
Re-preparation of quencher:
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 TEbuffer to get a stock of 50 uM in 20 ul
3.
Took 2 ul of 50 uM solution and added 98 ul of TE buffer to get a concentration of 1uM in 100 ul
NEB CRISPR + re-diluted 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.
Miniprep 4 HbcAg Innoculations:
Miniprep procedures for each innoculation:
1. Pellet 5ml bacterial overnight culture by centrifugation at 7830 rpm (6800 x g) for 3 min at room temperature (15–25°C).
2. Resuspend
pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction
to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the
solution will
turn colorless.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 60 s and discard
the
flow-through,
7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB. Centrifuge for 60 s and discard the flow-through.
8. Wash the QIAprep spin column by adding 0.75 ml Buffer PE. Centrifuge for 60
s and discard the flow-through.
9. Centrifuge for 1 min to remove residual wash buffer.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5)
or
water to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
THURSDAY : 10/03/2019
Nano-drop of HbcAg Minipreps from yesterday:
SUNDAY : 10/06/2019
HbcAg Miniprep 4 Serial Dilutions
PCR of minipreped plasmid pcaA
Resuspension of Lyophilized Primers
a. Added volume of water indicated on order sheet (differed for each primer) to make 100 μM of each primer
b. Pipetted up and down to mix
Preparation of PCR mix
a.
Prepared 10 μM working stock of each primer by extracting 5 ul of 100 uM stock and adding 45 ul of distilled water to a
total of 50 ul.
b. Labelled each eppendorf tube with name of gene and sample number (1,2,3, postive control,
negative control)
c. Added 20 ul PCR Mastermix to each tube
d. Added 1 ul of 10 uM forward primers to each appropriate tube. This was done for all genes.
e. Added 1 ul of 10 uM reverse primers to each appropriate tube.
This was done for all genes.
f. Added 2ul DNA minipreps (1, 2, 3, and gblock) to each tube. None was added to negative control.
g. Water was added to reach a total volume ot 40 ul (16 ul for each tube with DNA and 18 ul for
negative control)
h. PCR temperatures were set on machine according to BioRad protocol and 40 cycles were done.
i. PCR was left overnight.
Preparation of 16μl RPA+SYBR Green Mastermix + 10.75μl HbcAg 60ng/μl sample to be run on microfluidics chip
a. in one RPA reaction tube:
a. Primer A (10μM) - 2.4μL
b. Primer B (10μM) - 2.4μL
c. Rehydration
Buffer - 29.5μL
d. dH2O - 8.2μL
b. added 15μL each from tube to 2 PCR tubes
c. added 1μL dilluted SYBR Green to the 2 PCR tubes
d. In 10μL 60ng/μL DNA sample and 10μL negative control, 0.75 μL Magnesium Acetate was
added to each.
e. The tubes were given to the engineering team.
MONDAY : 10/07/2019
Serial dilution of is481 mp2
HbcAg PCR with optimized annealing temperature
1. Added 20 ul PCR Mastermix to each PCR tube
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. Annealing temperature was set
to slightly below Tm of the primers. (61 degrees)
7. PCR temperatures were set on machine according to BioRad protocol and 35 cycles were done.
Gel Electrophoresis of Optimized HbcAg RPA
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
SYBR Green Verification of Optimized HbcAg RPA
1. 0.8μl of diluted SYBR Green was added to each tube.
2. The reactions were placed under UV Lamp at 384 nm to observe fluorescence.
TUESDAY : 10/08/2019
Serial Dilution of ypo2088 minprep 1
15μl RPA with 10μl sample DNA (done with ypo2088)
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 20 min using thermocycler
THURSDAY : 10/10/2019
Cra PCR
I. Reaction mix in 1. PCR tube:
a. Forward Primer (10μM) - 1.25μL
b. Reverse Primer (10μM) - 1.25μL
c. 2x Mastermix - 12.5μL
d. dH2O - 10μL
II. In Tube 1: Dislodged E.coli colony
from IS481 plate using pipette tip
III. Tube 2: -ve control
IV. Followed New England BioLabs protocal in setting run parameters
V. PCR left to run
SYBR Green Verification of Optimized HbcAg RPA
1. 0.8μl of diluted SYBR Green was added to each tube.
2. The reactions were placed under UV Lamp at 384 nm to observe fluorescence.
SYBR Green Verification of Optimized HbcAg RPA
1. 0.8μl of diluted SYBR Green was added to each tube.
2. The reactions were placed under UV Lamp at 384 nm to observe fluorescence.
SYBR Green Verification of Optimized HbcAg RPA
1. 0.8μl of diluted SYBR Green was added to each tube.
2. The reactions were placed under UV Lamp at 384 nm to observe fluorescence.
