Daniel:Notebook/ComboLock/2016-8-30
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X3 Dilution Test (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
- 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
- 20160830-X3Dilution-qPCR-Average.png
qPCR Data averaged across technical replicates
- 2016-08-30-X3Dilutions-0-2.png
Gel Image 0-2
- 2016-08-30-X3Dilutions-3-5.png
Gel Image 3-5
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+11 | A1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 0B | 1.00E+11 | B1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 0C | 1.00E+11 | C1 | 34 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1A | 1.00E+10 | D1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1B | 1.00E+10 | E1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1C | 1.00E+10 | F1 | 35 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2A | 1.00E+09 | G1 | 36 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2B | 1.00E+09 | H1 | 36 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2C | 1.00E+09 | A12 | 36 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3A | 1.00E+08 | B12 | 37 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3B | 1.00E+08 | C12 | 37 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3C | 1.00E+08 | D12 | 37 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4A | 1.00E+07 | E12 | 38 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4B | 1.00E+07 | F12 | 38 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4C | 1.00E+07 | G12 | 38 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5A | 1.00E+06 | H12 | 39 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5B | 1.00E+06 | A2 | 39 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5C | 1.00E+06 | B2 | 39 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | C2 | 41 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |