Daniel:Notebook/ComboLock/2016-10-4

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Primer24 qPCR Test (From Yesterday)[edit]

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Continuing the primer amplifications to test off-target binding.

Protocol[edit]

  1. qPCR
    1. Set up qPCR reactions according to table
    2. Sample Molecule Count Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM primer24 100 uM AmpF H2O Total Volume (uL)
      Sample 0A 1.00E+09 A1 2 25 0.5 0.5 22 50
      Sample 0B 1.00E+09 A2 2 25 0.5 0.5 22 50
      Sample 0C 1.00E+09 A3 2 25 0.5 0.5 22 50
      NTC (rev) 0 A4 0 25 0.5 0.5 22 50
    3. Make the following master mix
      1. 90.2 uL nfH2O
      2. 2.05 uL 100 uM primer12 RC
      3. 2.05 uL 10 uM Amp6.3R Index 30
      4. 102.5 uL Kapa SYBR Fast
    4. Aliquot 43 uL MM1 into each well
    5. Add 2 uL sample to appropriate well
    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 x26
      7. 72C 2 min
      8. 16C hold
  2. TBE Gel
    1. Mix 48 uL TBE, 12 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 22 minutes at 250V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results[edit]