Daniel:Notebook/ComboLock/2017-6-19

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Revision as of 19:25, 19 June 2017 by >Djacobse (→‎Library Prep)
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Production Run (Started Wednesday 5/8)

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Library Prep

  1. Qiaquick Purification
    1. Combine 20 uL each sample into a 1.5 mL tube following matrix
    2. Add 200 uL (5X) PB (binding buffer) to sample
    3. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    4. Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
    5. Repeat above step with 400 uL PE buffer
    6. Dry spin column for 2 minutes at 14000 rpm; discard flow through
    7. Let stand with cover open in fume hood for ~5 minutes
    8. Transfer column to a new 1.5 mL eppendorf tube
    9. Add 30 uL nfH2O to column
    10. Spin for 1 minute at 14000 rpm
  2. Size Select TBE Gel
    1. Mix 30 uL 10X TBE and 24 uL 6X dye
    2. Aliquot 9 uL for each sample/ladder into a separate tube
    3. Add 15 uL sample or 2.5/12.5 uL ladder/10X TBE to aliquot tubes
    4. Mix and add 23 uL per lane to gel
    5. Run gel at 230V for 24 minutes
    6. Add 2 uL SYBR gold and incubate on shaker for 3 minutes
    7. Rinse and image in gel doc
    8. Cut out bands at approximately 221bp
    9. Add extracted gel bands to 0.5mL tube with 0.2 gauge needle hole inside a 1.5 mL tube
    10. Centrifuge for 1:30 at 13000rpm
    11. Throw away 0.5mL tube and add 500 uL TE buffer to each tube
    12. Incubate at 37C for 1 hour
    13. Centrifuge for 1:30 at 13000rpm
    14. Withdraw supernatant and add it to a nanosep column
    15. Centrifuge for 1:30 at 13000 rpm
    16. Remove flow through and add it to new 2 mL tube (~500 uL)
  3. Ethanol Precipitation
    1. Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1 uL glycoblue to each tube
    2. Incubate overnight at -80C

Continued tomorrow