Daniel:Notebook/ComboLock/2016-8-19
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Positive Control Amplicon Test (Started yesterday)
Protocol
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 15 uL exonuclease I (20 units/μL) and 3 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
- Elution
- Add 25 uL prewarmed elution buffer; vortex to suspend
- Incubate at RT for 2 minutes
- Apply magnet and transfer supernatant to a clean centrifuge tube
- Repeat elution with 25 uL fresh elution buffer; add supernatant to first elution
- qPCR
- Set up reaction according to table
- Master Mix
- 227.5 uL KAPA SyberFast MM
- 159.25 uL nfH2O
- 4.55 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 | Name | 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 1 | Amp1 Test | A1 | 10 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | Amp3 Test | B1 | 11 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | Latch X2 Specificity | C1 | 12 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | Latch X3 Specificity | D1 | 13 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | No Template | E1 | 14 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | No Latch | F1 | 15 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 7 | No Padlock | G1 | 16 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 8 | PCAmp2 Only | H1 | 17 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 9 | PCR NTC | A12 | 18 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |