Daniel:Notebook/ComboLock/2016-9-5
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X3 Dilution Test (Started Monday)[edit]
Protocol[edit]
- Size Select Gel
- For sample lanes
- Mix 10 uL from each replicate (30 total) in separate tubes for each sample
- Use lanes 1A-H and lane A12
- Add 6 uL 6X loading dye and 4 uL TBE for 40 uL total
- For sample 1, do this twice (80 uL total)
- For ladder lane mix 4 uL 25bp ladder, 6 uL 6X loading dye and 30 uL TBE (40 uL total)
- Add 40 uL to each lane
- 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 desired bands (see gel image) and put into tubes; 2 lanes per tube (2 tubes total)
- Image in gel doc post-extraction
- 2016-09-05-X3Dilution-SizeSelect.png
Before Image
- 2016-09-05-X3Dilution-SizeSelect-After.png
After Image
- Centrifuge tubes at 14000rpm 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 14000 rpm for 1.5 minutes
- Extract the supernatant and place into NanoSep column
- Centrifuge at 14000 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