Daniel:Notebook/PosSequencing/2017-5-24

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Revision as of 15:17, 25 May 2017 by >Djacobse (→‎Protocol)
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Barcode Oligo Production

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Making more double stranded barcode oligo since I'm running out. I'll make both EcoRI and Uracil versions.

Protocol

  1. qPCR
    1. Make the following master mixes
    2. Reagent uL Single rxn Master Mix Eco1 (8.2X) Master Mix Eco2 (8.2X) Master Mix Eco3 (8.2X) Master Mix BioU1 (8.2X) Master Mix BioU2 (8.2X) Master Mix BioU3 (8.2X)
      Biotin-Uracil Primer (10 uM) 1 0 0 0 8.2 8.2 8.2
      Biotin-EcoRI Primer (10 uM) 1 8.2 8.2 8.2 0 0 0
      Primer 2 (10 uM) 1 8.2 8.2 8.2 8.2 8.2 8.2
      Template Oligo (100 pM) 2 16.4 16.4 16.4 16.4 16.4 16.4
      Kapa 2X Master Mix 25 205 205 205 205 205 205
      nfH2O 21 172.2 172.2 172.2 172.2 172.2 172.2
      Total 50 410 410 410 410 410 410
    3. Add 50 uL (dual) to the appropriate lanes
    4. File:PlateLayout-20170520-SinglePrimer.png
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold