Daniel:Notebook/ComboLock/2016-9-2
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X3 Dilution Test (Monday)[edit]
After talking with Dr. Zhang today, I'm going to sequence the bad products from Tuesday. This will hopefully elucidate the issues with the unwanted products, and can help in primer design for reducing the "noise" associated with amplifying these off-target products.
Protocol[edit]
- Size Select Gel
- For sample lanes, mix 10 uL each sample from lanes 1A-H and lane A12 (mix in one tube), 20 uL 6X loading dye and 10 uL TBE (120 uL total)
- For ladder lanes, mix 2 uL 25bp ladder, 5 uL 6X loading dye and 23 uL TBE (30 uL total)
- Add 30 uL to each lane (2 gels total, 1 per sample)
- Run gel for 25 minutes at 250V
- While running, prepare tubes by puncturing a 0.5 mL tube with a 0.22 gauge needle and putting inside a 1.5 mL tube
- Stain gel for 3 minutes with 3 uL SYBR gold
- Image in gel doc
- Extract bands at 104 bp and put into tubes; 2 lanes per tube (4 tubes total, 2 per sample)
- Image in gel doc post-extraction
- 2016-09-02-X3Dilution-SizeSelect.png
Before Image
- 2016-09-02-X3Dilution-SizeSelect-After.png
After Image
- Centrifuge tubes at 12000rpm for 1.5 minutes
- Discard 0.5 mL tube and resuspend in 500 uL TE buffer
- Incubate at 37C for 1 hour with vigorous shaker
- 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
- In a 2 mL tube, add 1250 uL 100% EtOH, 50 uL 3 M NaOAc, and 2 uL glycoblue to 500 uL sample
- Incubate at -80C overnight; continued tomorrow