Daniel:Notebook/ComboLock/2017-1-25

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Revision as of 17:39, 25 January 2017 by >Djacobse (→‎Results)
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Circularization Test (Started Monday)

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Protocol-New Amplification Primers

These are new primers I designed. They are slightly adjusted from the locations of AmpF/AmpR. Hopefully there is less noise.

  1. qPCR
    1. Make qPCR master mix according to following recipe
      1. 361.2 uL nfH2O
      2. 17.2 uL AmpF-CLv3
      3. 17.2 uL AmpR-CLv3
      4. 430 uL Kapa SYBR Fast
    2. Add 48 uL master mix to each well
    3. Add 2 uL sample according to table
    4. Sample Product Amt Pre/Post RCA Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 0A 1pmol Pre A1 2 25 1 1 21 50
      Sample 0B 1pmol Pre A2 2 25 1 1 21 50
      Sample 2A 10 fmol Pre A3 2 25 1 1 21 50
      Sample 2B 10 fmol Pre A4 2 25 1 1 21 50
      Sample 4A 100 amol Pre A5 2 25 1 1 21 50
      Sample 4B 100 amol Pre A6 2 25 1 1 21 50
      Sample 6A 1 amol Pre A7 2 25 1 1 21 50
      Sample 6B 1 amol Pre A8 2 25 1 1 21 50
      Sample 0AX 1pmol Post H1 2 25 1 1 21 50
      Sample 0BX 1pmol Post H2 2 25 1 1 21 50
      Sample 2AX 10 fmol Post H3 2 25 1 1 21 50
      Sample 2BX 10 fmol Post H4 2 25 1 1 21 50
      Sample 4AX 100 amol Post H5 2 25 1 1 21 50
      Sample 4BX 100 amol Post H6 2 25 1 1 21 50
      Sample 6AX 1 amol Post H7 2 25 1 1 21 50
      Sample 6BX 1 amol Post H8 2 25 1 1 21 50
    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 x24
      7. 72C 2 min
      8. 16C hold
  2. TBE Gel
    1. Mix 80 uL TBE, 20 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