Daniel:Notebook/ComboLock/2016-8-17
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Positive Control Amplicon Production (Started yesterday)[edit]
Protocol[edit]
- Size select gel
- Mix 30 uL TBE, 6 uL loading dye, and 4 uL sample for C1-C2 and C3-C2
- Mix 15 uL TBE, 3 uL loading dye, and 2 uL ladder
- Add 20 uL to each lane (2 lanes for C1-C2 and C3-C2)
- Run the gel at 250V for 25 min
- Stain with 3 uL SYBR gold for 3 min
- Image, and extract bands between 100bp and 125 bp; collect the gel bands in the same tube
- 2016-08-17-PosControlAmplicon-SizeSelect.png
Before Image
- 2016-08-17-PosControlAmplicon-SizeSelect-After.png
After Image
- Shred the gel by centrifuging at 14000rpm for 1 min 30 sec
- Incubate in 500 uL TBE for 1 hour at 37C with vigorous shaking
- Centrifuge at 12000rpm for 1.5 min
- Extract supernatant and run through a nanosep column; centrifuge for 1.5 min at 12000rpm
- Ethanol Precipitation
- Move flow through to a 2 mL tube
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue
- Incubate at -80C for 4 hours
- Pellet by centrifuging for 25 min at 12000rpm at 4C
- Remove supernatant and add 750 uL chilled 70% EtOH
- Centrifuge for 12 min at 12000 rpm and 4 C
- Remove supernatant and air dry
- Resuspend in 30 uL nfH2O and measure in nanodrop
Nanodrop Results[edit]
Sample | ng/uL ssDNA |
PCAmp1 (C1-C2) | 18.1 |
PCAmp3 (C3-C2) | 16.0 |