Daniel:Notebook/ComboLock/2017-5-2

From ZhangLabWiki
Revision as of 18:01, 2 May 2017 by >Djacobse (→‎Library Prep)
Jump to navigation Jump to search

Production Run (Started Friday 4/28)

Back to Calendar

Library Prep

  1. Qiaquick Purification
    1. Combine 20 uL each sample into a 1.5 mL tube following matrix
    2. Tube Sample 1 Sample 2 Indexes
      1X 1AX 1BX 20
      5AX 5AX nfH2O 24
      1Y 1AY 1BY 25
      5AY,4AY 5AY 4AY 26,27
    3. Add 200 uL (5X) PB (binding buffer) to sample
    4. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    5. Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
    6. Repeat above step with 400 uL PE buffer
    7. Dry spin column for 2 minutes at 14000 rpm; discard flow through
    8. Let stand with cover open in fume hood for ~5 minutes
    9. Transfer column to a new 1.5 mL eppendorf tube
    10. Add 30 uL nfH2O to column
    11. 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