Daniel:Notebook/ComboLock/2016-10-11

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Padlock0301 Test (Started Friday 10-7)[edit]

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Repeating the qPCR performed on Saturday. That run failed. I learned later that I had made an error in the amplification primer (I was using 2 reverse primers). I designed and ordered AmpF-CL-v2, which should be correct.

Protocol[edit]

  1. qPCR
    1. Set up reaction according to table in next section
    2. Make master mixes according to following recipes
      1. 264.25 uL nfH2O
      2. 377.5 uL SYBR Fast
      3. 7.55 uL 100 uM AmpF-CL-v2
    3. Add 43 uL master mix to each well
    4. 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 144 uL TBE, 36 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

qPCR Sample Table[edit]

Sample Template Molecule Count Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM AmpF-CL-v2 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
Sample 1A 10 pmol A1 20 2 25 0.5 5 17.5 50
Sample 1B 10 pmol B1 20 2 25 0.5 5 17.5 50
Sample 1C 10 pmol C1 20 2 25 0.5 5 17.5 50
Sample 2 None D1 21 2 25 0.5 5 17.5 50
Sample 3 10 pmol E1 22 2 25 0.5 5 17.5 50
Sample 4A 100 fmol F1 23 2 25 0.5 5 17.5 50
Sample 4B 100 fmol G1 23 2 25 0.5 5 17.5 50
Sample 4C 100 fmol H1 23 2 25 0.5 5 17.5 50
Sample 5A 1 fmol A12 24 2 25 0.5 5 17.5 50
Sample 5B 1 fmol B12 24 2 25 0.5 5 17.5 50
Sample 5C 1 fmol C12 24 2 25 0.5 5 17.5 50
Sample 6A 10 amol D12 27 2 25 0.5 5 17.5 50
Sample 6B 10 amol E12 27 2 25 0.5 5 17.5 50
Sample 6C 10 amol F12 27 2 25 0.5 5 17.5 50
NTC 0 G12 28 0 25 0.5 5 19.5 50

Results[edit]