Daniel:Notebook/ComboLock/2016-9-30
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AmpR Test
Yesterday I tested the AmpR primer (the names are actually confusing, the name is the primer I'm NOT using. So yesterday I amplified off the circularized product using AmpR (which binds to the LinkerL region of the circularized product) and primer12RC, which binds to the amplified product. Now I'm going to test the amplification of using the AmpF primer. If AmpF is the culprit (which is what I expect) then I should see off-target binding. If the off-target products require both AmpF and AmpR, I shouldn't see off-target product.
Protocol
- qPCR
- Follow reaction table below to set up 4 reactions
- Make the following master mix
- 90.2 uL nfH2O
- 2.05 uL Primer2
- 2.05 uL AmpF6.4Sol
- 102.5 uL SYBR Fast
- Aliquot 48 uL master mix into each well
- Add 2 uL sample to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x26
- 72C 2 min
- 16C hold
- 20160930-qPCRCurve-Primer2.png
qPCR curve
- 2016-09-30-qPCR-Primer2.png
Gel image
Sample | Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM AmpF6.4Sol | 100 uM primer2 | H2O | Total Volume (uL) |
Sample 0A | 1.00E+10 | A1 | NA | 2 | 25 | 0.5 | 0.5 | 22 | 50 |
Sample 0B | 1.00E+10 | A2 | NA | 2 | 25 | 0.5 | 0.5 | 22 | 50 |
Sample 0C | 1.00E+10 | A3 | NA | 2 | 25 | 0.5 | 0.5 | 22 | 50 |
NTC | 0 | A4 | NA | 0 | 25 | 0.5 | 0.5 | 24 | 50 |