Daniel:Notebook/ComboLock/2016-10-8
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Padlock0301 Test (Started yesterday)
Protocol
- 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
- qPCR
- Set up reaction according to table in next section
- Make master mixes according to following recipes
- 334.25 uL nfH2O
- 477.5 uL SYBR Fast
- 9.55 uL AmpF
- 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 x24
- 72C 2 min
- 16C hold
qPCR Sample Table
Sample | Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF-CL | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1A | 1.00E+09 | A4 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1B | 1.00E+09 | A5 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1C | 1.00E+09 | A6 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | 1.00E+10 | A7 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | 1.00E+09 | H2 | 27 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4A | 1.00E+10 | H5 | 28 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4B | 1.00E+10 | H6 | 28 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4C | 1.00E+10 | H7 | 28 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5A | 1.00E+09 | H8 | 29 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5B | 1.00E+09 | D1 | 29 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5C | 1.00E+09 | D2 | 29 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6A | 1.00E+09 | H8 | 30 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6B | 1.00E+09 | D1 | 30 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6C | 1.00E+09 | D2 | 30 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | D3 | 31 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |