Daniel:Notebook/PosSequencing/2017-7-11

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Revision as of 17:43, 11 July 2017 by >Djacobse (→‎PolyT Primer qPCR)
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emPCR8 (Started Thursday July 6)

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PolyT Primer qPCR

The purpose of this slight deviation is to see if using the PolyT primer (which adds another 22 bases) to the final sequence produces an NTC band that is distinct from the product band.

  1. qPCR
    1. Dilute 2 uL each 10 uM 2-Biotin-Uracil and Primer2 into 16 uL nfH2O (1:10)
    2. Prepare the following 4.2X (1X) master mix
      1. 16.8 uL 1 uM primers (4, 2 each primer; conc 0.05 uM)
      2. 58.8 (16) uL nfH2O
      3. 84 uL 2X Kapa SYBR master mix
    3. Aliquot 38 uL master mix into each lane
    4. Add 2 uL appropriate sample according to plate layout
    5. File:PlateLayout20170711-emPCR8-PolyT.png
    6. Run the single primer protocol (main difference is 45C melting temp
    7. for ~40 cycles
  2. TBE Gel
    1. Mix together 64 uL 1X TBE and 16 uL 6X loading dye
    2. Aliquot 10 uL per sample onto parafilm
    3. Add 1.5 uL 25bp ladder or 2 uL sample to appropriate lanes
    4. Mix and add 10 uL to appropriate gel wells
    5. Run gel for 25 minutes at 235V
    6. Open gel and stain with 2 uL SYBR gold for 3 minutes
    7. Rinse once and image in gel doc
  3. Run the single primer protocol (main difference is 45C melting temp
  4. for ~40 cycles

    qPCR Results

    <gallery perrow=2 heights=250px widths=350px mode=packed-hover> File:PlateLayout20170711-emPCR8-PolyT.png|Plate layout File:|Plate CTs File:|Raw curves </gallery