Daniel:Notebook/ComboLock/2016-10-1
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PCR Testing (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 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 0A | 1.00E+10 | A1 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 0B | 1.00E+10 | A2 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 0C | 1.00E+10 | A3 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
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 2A | 1.00E+10 | A7 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2B | 1.00E+10 | A8 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2C | 1.00E+10 | H1 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3A | 1.00E+09 | H2 | 27 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3B | 1.00E+09 | H3 | 27 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3C | 1.00E+09 | H4 | 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 |
NTC | 0 | D3 | 30 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |
Results
Note: I stopped the qPCR reaction for lanes A1-H1 (Samples 0A-C,1A-C,2A,2B) at cycle 24 since they had reached saturation.
- 20161001-qPCR-LatchX4Test1.png
qPCR Curve
- 2016-10-01-PCAmpX4Test.png
Gel Image 1
- 2016-10-01-PCAmpX4Test2.png
Gel Image 2
Discussion
Well, this is a mixed success.