Daniel:Notebook/PosSequencing/2017-6-9: Difference between revisions

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File:20170609-aqPCRStage2-plateCTs.png|Raw CT values
File:20170609-aqPCRStage2-plateCTs.png|Raw CT values
File:20170609-aqPCRStage2-plateCTs-normed.png|CT values normalized to NTC (Lane A6)
File:20170609-aqPCRStage2-plateCTs-normed.png|CT values normalized to NTC (Lane A6)
File:|Raw curves
File:20170609-aqPCRStage2-rawcurves.png|Raw curves
File:2017-06-09-aqPCR-beadFraction.png|Gel image-bead fraction
File:2017-06-09-aqPCR-beadFraction.png|Gel image-bead fraction
File:2017-06-09-aqPCR-Supernatant.png|Gel image-supernatant fraction
File:2017-06-09-aqPCR-Supernatant.png|Gel image-supernatant fraction

Revision as of 22:12, 9 June 2017

Aqueous PCR 2 (Started Tuesday)

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Protocol

  1. Supernatant Extraction
    1. Vortex qPCR lanes from Tuesday for several seconds
    2. Magnet pulldown for 1 minute; remove supernatant and keep, putting into new 0.2 mL tubes
  2. qPCR
    1. Make the following 6.2X (1X) master mix
      1. 142.6 (23) uL nfH2O
      2. 6.2 (1) uL 10 uM Biotin-Uracil primer
      3. 6.2 (1) uL 10 uM Primer 2
      4. 155 (25) uL Kapa 2X SYBR master mix
    2. Add 50 uL (resuspend beads) to each of the 6 lanes from Tuesday's qPCR following plate layout
    3. File:PlateLayout-20170609-aqPCRStage2.png
    4. Make the following 8.2X master mix
      1. 147.6 (18) uL nfH2O
      2. 8.2 (1) uL 10 uM Biotin-Uracil primer
      3. 8.2 (1) uL 10 uM Primer 2
      4. 205 (25) uL Kapa 2X SYBR master mix
    5. Add 45 uL master mix to each of the H lanes following plate layout
    6. Add 5 uL appropriate supernatant, 1 pM barcode 2 oligo, or nfH2O following plate layout
    7. Apply caps tightly, vortex 5 seconds and spin down
    8. Use following cycles for qPCR
        1. 95C 2 min
        2. 95C 20 sec
        3. 58C 30 sec
        4. 70C 30 sec
        5. Goto b 40 times
        6. 4C forever

    Results