Daniel:Notebook/ComboLock/2016-7-19
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C Probe Test 3-SOD1/VIM-Stringent Washes (Started yesterday)[edit]
Protocol[edit]
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 10 uL exonuclease I (20 units/μL) and 2 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
- Master Mix
- 302.5 uL KAPA SyberFast MM
- 192.5 uL nfH2O
- 6.05 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
- Stringent-ExpA-20160719.png
qPCR Curve; Experiment A
- 2016-07-19-Stringent-ExpA.png
Gel Image; Experiment A
- Stringent-ExpB-20160719.png
qPCR Curve; Experiment B
- 2016-07-19-Stringent-ExpB.png
Gel Image; Experiment B
Sample | Lane | AmpR Index | Cells | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1A | A1 | 21 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2A | B1 | 22 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3A | C1 | 23 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4A | D1 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5A | E1 | 25 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1B | F1 | 26 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2B | G1 | 27 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3B | H1 | 28 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4B | A12 | 29 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5B | B12 | 30 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | C12 | 31 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |