Daniel:Notebook/PosSequencing/2017-7-7

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emPCR8[edit]

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Protocol-Part 2[edit]

  1. 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; resuspend beads in the solution and take 15 uL aside for testing
  2. 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 30 minutes at 37C
    8. Heat inactivate by incubating for 20 minutes at 65C
  3. 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 50 uL 2XSSC
  4. Dye hybridization
    1. Aliquot 20 uL sample to a new tube
    2. Add 20 uL 4X SSC and 50% formamide
    3. Add 1 uL appropriate (10 uM) dye, A488-dc6 for sample 1 and Cy5-dc6 for sample 3
    4. Incubate in the dark (RT) for 15 minutes
    5. Magnet pulldown, remove supernatant and add 40 uL 2X SSC buffer
    6. Add 15 uL to a slide for analysis
    7. Seal coverslip with nail polish and wait approximately 30 minutes to dry
  5. Imaging Parameters
    1. Confocal Microscope
    2. 488 Laser-3.00%
      1. PMT Trans-Gain 225V
      2. PMT1-Range:500-550-Gain 550V
      3. PMT3-Range:750-800-Gain 600V
    3. 642 Laser-8.00%
      1. PMT Trans-Gain 300V
      2. PMT1-Range:500-550-Gain 6000V
      3. PMT3-Range:750-800-Gain 550V