Daniel:Notebook/PosSequencing/2017-6-27

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emPCR 6 (Started Yesterday)[edit]

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Protocol[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; resuspend beads in the solution and take 15 uL aside for testing
    ******
    
  3. Strand Separation (DNA Denaturation)
    1. Add 500 uL KOH buffer (pH 12.0)
    2. Spin down sample at 13,000rpm for 1 minute
    3. Remove all but 40uL buffer
    4. Repeat KOH steps A-C once more
    5. Add 500 uL 4X SSC buffer (pH 12.0)
    6. Spin down sample at 13,000rpm for 1 minute
    7. Remove all but 10uL buffer
    8. Resuspend in 80 uL 2X SSC buffer
  4. Single primer qPCR
    1. Make the following master mixes
    2. Reagent uL in Single Rxn Master Mix P2 (6.2X) Master Mix BioU (6.2X) Master Mix Dual (8.2X)
      Primer 2 (10 uM) 1 6.2 0 8.2
      BioU Primer (10 uM) 1 0 6.2 8.2
      2X Kapa SYBR Master Mix 20 124 124 164
      nfH2O 16 99.2 99.2 123
      Total* (+ 5 uL sample=40) 37 229.4 229.4 303.4
    3. Aliquot 37 uL master mix to appropriate lanes
    4. Add 3 uL sample according to plate layout below
    5. File:PlateLayout20170627-emPCR6-SinglePrimer.png
    6. Run the following thermocycler program
      1. 95C 3 min
      2. 95C 3 sec
      3. 45C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x120
      7. 72C 2 min
      8. 16C hold

Results[edit]

So I stopped this run early, because the NTC lane of the dual primer samples amplified prior to either of the single-stranded primer lanes. However, the dsDNA lanes for both the nM and pM samples amplified 5+ cycles prior to the NTC. So the emPCR actually worked and there is something on those beads! However, the single-stranded protocol took away too much DNA again, even though I dropped the pH to 11.5.