Daniel:Notebook/ComboLock/2017-3-27

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Revision as of 16:25, 27 March 2017 by >Djacobse (→‎Results)
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RCA Test (Started March 24)

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Protocol

  1. Rolling Circle Amplification
    1. Add 20 uL nfH2O to every sample
  2. qPCR
    1. Make master mixes according to following table
    2. Reagent 1X Rxn MM-Amp (33.2X) MM-Prod (9.2X)
      Primer Pair NA AmpF/AmpR p4/p12RC
      Forward Primer (10 uM) 1 33.2 9.2
      Forward Primer (10 uM) 1 33.2 9.2
      Kapa SYBR Fast Master Mix (2X) 25 830 230
      nfH2O 21 697.2 193.2
      Total 48 1593.6 441.6
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to plate layout
    5. File:PlateLayout-20170327-RCATest-CPLP.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
  3. Qubit Measurement
    1. Add 995 uL buffer and 5 uL ssdye to tube
    2. Add 190 uL buffer and 10 uL correct standard to standard tubes
    3. Add 199 uL buffer and 1 uL sample to sample tubes
    4. Let sit 5-10 minutes in the dark
    5. Measure in Qubit
  4. TBE Gel
    1. Mix 120 uL TBE and 40 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

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