Daniel:Notebook/ComboLock/2017-3-9

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Bolt Test (Started Yesterday)[edit]

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Protocol[edit]

  1. Bolt/Latch Hybridization
    1. Wash twice with 100 uL wash buffer
  2. Elution
    1. Add 50 uL wash buffer to each sample
    2. Cook at 95C for 15 minutes
  3. qPCR
    1. Make master mixes according to following recipes
    2.   Single Rxn MMC1 (11.2X) MMC2 (11.2X) MMP (12.2X)
      Primer Pair NA p24/p4RC p6/p12RC p24/p12RC
      uL Fwd Primer (10 uM) 1 11.2 11.2 12.2
      uL Rev Primer (10 uM) 1 11.2 11.2 12.2
      uL nfH2O 21 235.2 235.2 256.2
      uL Kapa SYBR Fast 25 280 280 305
      Total 48 537.6 537.6 585.6
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to plate layout
    5. File:PlateLayout-20170309-BoltTest.png
      • Note: The parentheses in the descriptions indicate which sample was used
      • Note:The colors are according to master mix, targeting either C1 (blue), C2 (red), or product (green)
    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
  4. TBE Gel
    1. Mix 360 uL TBE, 90 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 23 minutes at 230V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results[edit]