Daniel:Notebook/PosSequencing/2017-7-13

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emPCR 9-Concentration Comparison (Started Yesterday)[edit]

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Protocol-Part 2[edit]

  1. Prepare Water saturated compounds
    1. Mix 5 mL diethyl ether and 5 mL ddH2O in a falcon tube; allow to settle, extract top (ether) phase
    2. Mix 5 mL ethyl acetate and 5 mL ddH2O in a falcon tube; allow to settle, extract top (acetate) phase
  2. Emulsion Breaking
    1. With a 200 uL pipette add 100 uL diethyl ether to each sample; save the tip
    2. Adjust tip to 200 and using same tip pool samples in a new 1.5 mL tube
    3. Vortex the pooled sample in the 1.5 mL tube for 30 seconds
    4. Spin down the sample 13,000 rpm for 5 minutes
    5. Take off oil-DE mix (top layer) but do not disrupt the oil/aqueous interface
    6. Add 1000 uL ethyl acetate and vortex for 30 seconds
    7. Spin down sample at 13,000rpm for 3 minutes
    8. Remove ethyl acetate without disturbing the pellet
    9. Add 1000 uL diethyl ether and vortex for 30 seconds
    10. Spin down sample at 13,000rpm for 3 minutes
    11. Remove DE without disturbing the aqueous phase
    12. Allow DE to dry in the fume hood
    13. Resuspend beads in the solution and split in half for testing (40-50 uL)
  3. Phosphorylation
    1. Pull down beads, remove supernatant, and wash with 100 uL DynaT-buffer (Dynabuffer + 0.05% Tween20)
    2. Pull down beads and remove supernatant
    3. Resuspend the beads in the following mixture
    4. Reagent uL 1X Reaction uL 3.2X Master Mix
      T4 PNK Buffer (10X) 5 16
      ATP (10 mM) 5 16
      T4 PNK (10 U/uL) 1 3.2
      nfH2O 39 124.8
      Total 50 160
    5. Resuspend each sample in 49 uL master mix
    6. Add 1 uL T4 PNK to each
    7. Incubate for 1 minutes at 37C
    8. Heat inactivate by incubating for 20 minutes at 65C
  4. Lambda Exonuclease Digestion
    1. Pull down with magnet and remove supernatant
    2. Make the following master mix
      1. 16 uL 10X Lambda exonuclease reaction buffer
      2. 140.8 uL nfH2O
    3. Resuspend beads in 49 uL master mix
    4. Add 1 uL lambda exonuclease (10 U/uL)
    5. Incubate for 30 minutes at 37C
    6. Heat kill enzyme by incubating for 10 minutes at 80C
    7. Magnet pulldown, and resuspend in 40 uL 2XSSC

Continued tomorrow