Daniel:Notebook/PosSequencing/2017-7-10: Difference between revisions

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So there was plenty of material ''after emulsion'' but it was mostly lost during the PNK/lambda reactions
So there was plenty of material ''after emulsion'' but it was mostly lost during the PNK/lambda reactions
[[Category:PosSeq]] [[Category:20170706]]

Revision as of 15:38, 11 July 2017

emPCR8 (Started Thursday July 6)

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Dye Hybridization and Imaging

Since Saturday's results showed nothing, I'm going to try again but with the protocol the same as the first Lambda exonuclease test

  1. Dye hybridization
    1. Mix 20 uL ssDNA (lambda digested) and 20 uL 2X SSC
    2. Add 1 uL appropriate (10 uM) dye, A488-dc6 for sample 1 and Cy5-dc6 for sample 3
    3. Incubate in the dark (RT) for 15 minutes
    4. Magnet pulldown, remove supernatant and add 40 uL 2X SSC buffer
    5. Add 15 uL to a slide for analysis
    6. Seal coverslip with nail polish and wait approximately 30 minutes to dry

    Results showed nothing. I'll have to do qPCR to see if there was anything there to begin with.

  2. emPCR8
    1. Dilute 2 uL each 10 uM 2-Biotin-Uracil and Primer2 into 16 uL nfH2O (1:10)
    2. Prepare the following 7.2X (1X) master mix
      1. 14.4 uL 1 uM primers (2, 1 each primer)
      2. 115.2 (16) uL nfH2O
      3. 144 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:PlateLayout20170710-emPCR8-benchmarks.png

      qPCR Results

      <gallery perrow=2 heights=250px widths=350px mode=packed-hover> File:PlateLayout20170710-emPCR8-benchmarks.png|Plate layout File:EmPCR8-20170710-benchmarks-plateCTs-normed.png|Plate CTs File:EmPCR8-20170710-benchmarks-rawcurves.png|Raw curves </gallery

      So there was plenty of material after emulsion but it was mostly lost during the PNK/lambda reactions