Daniel:Notebook/ComboLock/2016-9-23
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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
Control
Standard qPCR like I normally do. Control version for the tests after.
- 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
- 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
Sample | Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1A | 1.00E+10 | A1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1B | 1.00E+10 | B1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1C | 1.00E+10 | C1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2A | 1.00E+09 | D1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2B | 1.00E+09 | E1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2C | 1.00E+09 | F1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | A1 | 36 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |
Primer Concentration