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

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<gallery perrow=2 heights=250px widths=350px mode=packed-hover>
<gallery perrow=2 heights=250px widths=350px mode=packed-hover>
File:|Plate Layout
File:PlateLayout-20170609-aqPCRStage2.png|Plate Layout
File:|Raw CT values
File:|Raw CT values
File:|CT values normalized to NTC (Lane A6)
File:|CT values normalized to NTC (Lane A6)
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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
File:2017-06-09-aqPCR-beadFraction-BAKE.png|Gel image-bead fraction post 95C bake (12 min)
</gallery>
</gallery>




[[Category:PosSeq]] [[Category:20170606]]
[[Category:PosSeq]] [[Category:20170606]]

Revision as of 21:02, 9 June 2017

Aqueous PCR 2 (Started Tuesday)

Back to Calendar

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