Daniel:Notebook/ComboLock/2016-8-29
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Positive Control Dilution Test (Started Friday)
Protocol
Today I'm testing the samples using the P2 and P12RC amplification primers. Yesterday included the original amplicon materials; this qPCR should be only the DNA still attached to beads.
- qPCR X2-Primers
- Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC
- Load lanes according to table
- Master Mix
- 352.5 uL KAPA SyberFast MM
- 310.2 uL nfH2O
- 7.05 uL primer2
- 7.05 uL primer12RC
- Add 48 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x32
- 72C 2 min
- 16C hold
- TBE Gel
- Mix together 72 uL TBE and 18 uL 6X loading dye in a 0.2mL tube
- Add 1 or 2 uL sample according to table
- Add 10 uL mix to well
- Run gel for 25 minutes at 250V
- Stain for 3 min with 3 uL SYBR gold
- Image in gel doc
Sample Name | Sample No. | qPCR Lane |
Pure C1-C2 | Sample 00 | A1 |
1E+11 | Sample 0 | B1 |
1E+10 | Sample 1 | C1 |
1E+09 | Sample 2 | D1 |
1E+08 | Sample 3 | E1 |
1E+07 | Sample 4 | F1 |
1E+06 | Sample 5 | G1 |
1E+05 | Sample 6 | H1 |
10000 | Sample 7 | A12 |
1000 | Sample 8 | B12 |
100 | Sample 9 | C12 |
10 | Sample 10 | D12 |
1 | Sample 11 | E12 |
NTC | NTC | F12 |
Gel Lane | Sample Name | qPCR Lane | uL Sample |
2 | 25 bp Ladder | 2 | |
3 | 1E11 Molecule | B1 | 1 |
4 | 1E9 Molecule | D1 | 1 |
5 | 1E7Molecule | F1 | 2 |
6 | 1E5 Molecule | H1 | 2 |
7 | 1000 Molecule | B12 | 2 |
8 | 10 Molecule | D12 | 2 |
9 | 1 Molecule | D13 | 2 |