Daniel:Notebook/PosSequencing/2017-6-29

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Revision as of 19:48, 30 June 2017 by >Djacobse (→‎qPCR Round 2)
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emPCR 7-Strand Separation (Started Yesterday)

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Protocol (From Yesterday)

  1. Strand Separation (DNA Denaturation)
    1. Split each sample (1 pM and 100 pM) into 4 parts-> 25 uL each-> put them in 1.5mL tubes
    2. Add 200 uL KOH buffer to each sample(pH 12.0, 11.5, 11.0, 10.0); denaturation pH of DNA should be around 11.3
    3. Spin down sample at 13,000rpm for 1 minute
    4. Remove all but 40uL buffer
    5. Repeat KOH steps A-C once more
    6. Add 200 uL 4X SSC buffer (pH 12.0)
    7. Spin down sample at 13,000rpm for 1 minute
    8. Remove all but 10uL buffer
    9. Resuspend in 80 uL 2X SSC buffer
  2. Single primer qPCR
    1. Make the following master mixes
    2. Reagent uL in Single Rxn Master Mix P2 (12.2X) Master Mix BioU (12.2X) Master Mix Dual (14.2X)
      Primer 2 (10 uM) 1 12.2 0 14.2
      BioU Primer (10 uM) 1 0 12.2 14.2
      2X Kapa SYBR Master Mix 20 244 244 284
      nfH2O 9 109.8 109.8 113.6
      Total* (+ 5 uL sample=40) 35 366 366 426
      • Notes: positive controls are 5 uL sample/5 uL nfH2O
    3. Aliquot 30 uL master mix to appropriate lanes
    4. Add 10 uL sample according to plate layout below
    5. File:PlateLayout20170629-emPCR7-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 x90
      7. 72C 2 min
      8. 16C hold

Results

qPCR Round 2

This is to double check the results of the previous experiment, which show little product for any the samples. Specifically,the post-emulsion breaking samples showed no discernible product, even though there was product in the pH 10 sample post-alkaline denaturation.

  1. Single primer qPCR
    1. Make the following 8.2X master mix
      1. 8.2 uL 10 uM primer2
      2. 8.2 uL 10 uM 2BiotinUracil
      3. 164 uL 10 uM 2X Kapa SYBR Fast master mix
      4. 131.2 uL 10 uM nfH2O
    2. Aliquot 38 uL master mix to appropriate lanes
    3. Add 2 uL sample according to plate layout below
    4. File:PlateLayout20170629-emPCR7-aqueousfractions.png
    5. 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 x90
      7. 72C 2 min
      8. 16C hold

Results