Daniel:Notebook/ComboLock/2016-9-2

From ZhangLabWiki
Jump to navigation Jump to search

X3 Dilution Test (Monday)[edit]

Back to Calendar

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]

  1. Size Select Gel
    1. 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)
    2. For ladder lanes, mix 2 uL 25bp ladder, 5 uL 6X loading dye and 23 uL TBE (30 uL total)
    3. Add 30 uL to each lane (2 gels total, 1 per sample)
    4. Run gel for 25 minutes at 250V
    5. 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
    6. Stain gel for 3 minutes with 3 uL SYBR gold
    7. Image in gel doc
    8. Extract bands at 104 bp and put into tubes; 2 lanes per tube (4 tubes total, 2 per sample)
    9. Image in gel doc post-extraction
    10. Centrifuge tubes at 12000rpm for 1.5 minutes
    11. Discard 0.5 mL tube and resuspend in 500 uL TE buffer
    12. Incubate at 37C for 1 hour with vigorous shaker
    13. Centrifuge at 12000 rpm for 1.5 minutes
    14. Extract the supernatant and place into NanoSep column
    15. Centrifuge at 12000 rpm for 1.5 minutes
  2. Ethanol Precipitation
    1. In a 2 mL tube, add 1250 uL 100% EtOH, 50 uL 3 M NaOAc, and 2 uL glycoblue to 500 uL sample
    2. Incubate at -80C overnight; continued tomorrow