Daniel:Notebook/ComboLock/2016-9-23: Difference between revisions
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===Annealing Temperature=== | ===Annealing Temperature=== |
Revision as of 19:14, 23 September 2016
PCR Testing (Started Yesterday
Protocol (all)
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 20 uL exonuclease I (20 units/μL) and 4 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
Standard qPCR like I normally do. Control version for the tests after.
- qPCR
- Follow reaction map below
- Note:"X"=5 uL primer per reaction; "O" indicates 1 uL primer
- Note:"TN" is a range of temperatures (gradient) from 50C (Lane 1) to 65C (Lane 12)
- Set up reaction according to table in next section
- Make master mixes according to following table
- Add 48 uL correct master mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- qPCR
- Set up reaction according to table
- Master Mix
- 480 uL KAPA SyberFast MM
- 336 uL nfH2O
- 9.5 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- qPCR
- Set up reaction according to table
- Master Mix
- 480 uL KAPA SyberFast MM
- 336 uL nfH2O
- 9.5 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
A | X,T1 | X,T2 | X,T3 | X,T4 | X,T5 | X,T6 | ||||||
B | X,T1 | X,T2 | X,T3 | X,T4 | X,T5 | X,T6 | ||||||
C | X,T1 | X,T2 | X,T3 | X,T4 | X,T5 | X,T6 | ||||||
D | O,T1 | O,T2 | O,T3 | O,T4 | O,T5 | O,T6 | ||||||
E | O,T1 | O,T2 | O,T3 | O,T4 | O,T5 | O,T6 | ||||||
F | O,T1 | O,T2 | O,T3 | O,T4 | O,T5 | O,T6 | ||||||
G | NTC | NTC | NTC | NTC | NTC | NTC | ||||||
H |
Master Mix | |||
Reagent | MMX | MMO | NTC |
PrimerF (100 uM) | 9.05 | 1.81 | 3.05 |
PrimerR (10 uM) | 90.5 | 18.1 | 30.5 |
KAPA Master Mix | 452.5 | 452.5 | 152.5 |
nfH2O | 316.75 | 396.39 | 118.95 |