Daniel:Notebook/ComboLock/2016-9-5

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X3 Dilution Test (Started Monday)[edit]

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Protocol[edit]

  1. Size Select Gel
    1. For sample lanes
      1. Mix 10 uL from each replicate (30 total) in separate tubes for each sample
      2. Use lanes 1A-H and lane A12
      3. Add 6 uL 6X loading dye and 4 uL TBE for 40 uL total
      4. For sample 1, do this twice (80 uL total)
    2. For ladder lane mix 4 uL 25bp ladder, 6 uL 6X loading dye and 30 uL TBE (40 uL total)
    3. Add 40 uL to each lane
    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 desired bands (see gel image) and put into tubes; 2 lanes per tube (2 tubes total)
    9. Image in gel doc post-extraction
    10. Centrifuge tubes at 14000rpm 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 14000 rpm for 1.5 minutes
    14. Extract the supernatant and place into NanoSep column
    15. Centrifuge at 14000 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