Daniel:Notebook/ComboLock/2017-3-17

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Circularization Comparison (T4 Poly/Ligase; Started Wednesday)[edit]

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Protocol-Part 6[edit]

  1. qPCR
    1. Make qPCR master mixes according to following recipe
    2. Reagent Single Rxn MM1 (23.2X) MM1-2 (20.2X) MM+ (C1, 4.2X)
      Primer Pair (fwd/rev) NA AmpF/AmpR-Ind21 AmpF/AmpR-Ind21 primer24/primer4*
      Forward Primer (10 uM) 1 23.2 20.2 4.2
      Reverse Primer (10 uM) 1 23.2 20.2 4.2
      nfH2O 21 487.2 424.2 88.2
      2X Kapa SYBR Fast 25 580 505 105
      Total 48 1113.6 969.6 201.6
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to plate layout
    5. File:PlateLayout-20170317-T4Circ-PostRCA.png
    6. 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
  2. TBE Gel
    1. Mix 200 uL TBE and 50 uL 6X dye
    2. Aliquot 10 uL per sample on to parafilm
    3. Add 2 uL sample or 1.5 uL ladder to aliquots
    4. Mix and add 10 uL per lane to gel
    5. Run gel at 230V for 24 minutes
    6. Add 2 uL SYBR gold and incubate on shaker for 3 minutes
    7. Rinse and image in gel doc

Results[edit]