Daniel:Notebook/ComboLock/2016-10-11
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Padlock0301 Test (Started Friday 10-7)[edit]
Repeating the qPCR performed on Saturday. That run failed. I learned later that I had made an error in the amplification primer (I was using 2 reverse primers). I designed and ordered AmpF-CL-v2, which should be correct.
Protocol[edit]
- qPCR
- Set up reaction according to table in next section
- Make master mixes according to following recipes
- 264.25 uL nfH2O
- 377.5 uL SYBR Fast
- 7.55 uL 100 uM AmpF-CL-v2
- 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
- TBE Gel
- Mix 144 uL TBE, 36 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
qPCR Sample Table[edit]
Sample | Template Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF-CL-v2 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1A | 10 pmol | A1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1B | 10 pmol | B1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 1C | 10 pmol | C1 | 20 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | None | D1 | 21 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | 10 pmol | E1 | 22 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4A | 100 fmol | F1 | 23 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4B | 100 fmol | G1 | 23 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4C | 100 fmol | H1 | 23 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5A | 1 fmol | A12 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5B | 1 fmol | B12 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5C | 1 fmol | C12 | 24 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6A | 10 amol | D12 | 27 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6B | 10 amol | E12 | 27 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6C | 10 amol | F12 | 27 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
NTC | 0 | G12 | 28 | 0 | 25 | 0.5 | 5 | 19.5 | 50 |
Results[edit]
- 20161011-qPCR-Padlock0301-AmpFCLv2.png
qPCR Curve
- 2016-10-11-Padlock0301-AmpFCLv2-Gel2.png
Gel Image 1
- 2016-10-11-Padlock0301-AmpFCLv2-Gel1.png
Gel Image 2