Daniel:Notebook/PosSequencing/2017-6-3

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Revision as of 18:28, 9 June 2017 by >Djacobse (→‎Results)
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Aqueous PCR Test (Started Yesterday)[edit]

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Protocol[edit]

  1. Wash Steps
    1. Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: A)
    2. Add 50 uL 4X SSC buffer to wash, pipette up and down to mix
    3. Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: B)
    4. Add 50 uL 4X SSC buffer to wash, pipette up and down to mix
    5. Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: C)
    6. Add 50 uL 4X SSC buffer to wash, pipette up and down to mix
    7. Pull down beads with magnet for 1 minute; remove and save supernatant (Fraction: D)
    8. Add 50 uL 4X SSC buffer to wash, pipette up and down to mix; Keep this fraction (Fraction: E)
  2. qPCR
    1. Make the following 14.2X master mix (1X)
      1. 14.2 uL 10 uM BiotinUracilPrimer1 (1)
      2. 14.2 uL 10 uM Primer2 (1)
      3. 284 uL 2X Kap SYBR Master Mix (20)
      4. 227.2 uL nfH2O (16)
    2. Aliquot 38 uL master mix to appropriate lanes
    3. Add 2 uL sample according to plate layout below
    4. File:PlateLayout-20170603-aqPCR.png
    5. Run the following thermocycler program
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x120
      7. 72C 2 min
      8. 16C hold

Results[edit]

Conclusions:

  • All the 1:4 ratio samples peak 1-3 cycles behind the full sample. Makes sense given that it should be 2
  • The H3 and H4 samples have too high of concentration. The H4 amplified in basically 0 samples and the H3 sample amplified even quicker, but the baseline correction really hurts the detection
  • There is basically nothing in any of the samples (at least above NTC baseline), but that unfortunately includes the elution fraction as well. It may require a 95C cook to denature them properly (technically 95C steps are in the qPCR also, but to elute I often use 10-15 minutes at 95C instead of like 3