Daniel:Notebook/ComboLock/2016-9-29
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Positive Control Amplicon Production (PCAmp4; Started yesterday)
Protocol
- Size Selection
- Centrifuge at 12000 rpm for 1.5 minutes
- Extract the supernatant and place into NanoSep column
- Centrifuge at 12000 rpm for 1.5 minutes
- Ethanol Precipitation
- Move sample to a fresh 2 mL tube
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc and 2 uL glycoblue
- Store at for 6 hours
- Pellet by centrifuging for 25 minutes at 12000rpm at 4C
- Remove supernatant and add 750 uL chilled 70% EtOH
- Centrifuge for 12 minutes at 12000rpm at 4C
- Remove supernatant and spin in vacuum centrifuge for 10 minutes
- Add 50 uL nfH2O and resuspend every tube in series;
- Measure final concentration in nanodrop
Results
Sample | ng/uL ssDNA |
C4-C2 Amplicon (Final) | 78.3 |
AmpF Test
Since I'm testing whether the AmpF primer (which has LinkerR that matches to primer2 and therefore has a potential off-target binding) creates the wrong product or not. To test this I'll be using the PCR primer Primer12-RC, which should be a forward primer for amplifying off the circular target. In conjuction with AmpR6.3, this should produce a 157bp product. Let's see what happens.
Protocol
- qPCR
- Follow reaction table below to set up 4 reactions
- Make the following master mix
- 71.75 uL nfH2O
- 2.05 uL Primer12RC
- 20.5 uL AmpR6.3-34
- 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
- 20160929-qPCRAmpFTest.png
qPCR Curve
- 2016-09-29-qPCR-Primer12RC.png
Gel Image