Daniel:Notebook/ComboLock/2016-8-29

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Revision as of 17:33, 29 August 2016 by >Djacobse (→‎Protocol)
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Positive Control Dilution Test (Started Friday)

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Protocol

Today I'm testing the samples using the P2 and P12RC amplification primers. Yesterday included the original amplicon materials; this qPCR should be only the DNA still attached to beads.

  1. qPCR X2-Primers
    1. Set up 14 reaction with 2 uL sample, 25 uL KAPA, 22 uL nfH2O, 0.5 uL primer2 and 0.5 uL primer12RC
    2. Load lanes according to table
    3. Sample Name Sample No. qPCR Lane
      Pure C1-C2 Sample 00 A1
      1E+11 Sample 0 B1
      1E+10 Sample 1 C1
      1E+09 Sample 2 D1
      1E+08 Sample 3 E1
      1E+07 Sample 4 F1
      1E+06 Sample 5 G1
      1E+05 Sample 6 H1
      10000 Sample 7 A12
      1000 Sample 8 B12
      100 Sample 9 C12
      10 Sample 10 D12
      1 Sample 11 E12
      NTC NTC F12
    4. Master Mix
      1. 352.5 uL KAPA SyberFast MM
      2. 310.2 uL nfH2O
      3. 7.05 uL primer2
      4. 7.05 uL primer12RC
    5. Add 48 uL Master Mix to each 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 x32
      7. 72C 2 min
      8. 16C hold
  2. TBE Gel
    1. Mix together 72 uL TBE and 18 uL 6X loading dye in a 0.2mL tube
    2. Add 1 or 2 uL sample according to table
    3. Gel Lane Sample Name qPCR Lane uL Sample
      2 25 bp Ladder   2
      3 1E11 Molecule B1 1
      4 1E9 Molecule D1 1
      5 1E7Molecule F1 2
      6 1E5 Molecule H1 2
      7 1000 Molecule B12 2
      8 10 Molecule D12 2
      9 1 Molecule D13 2
    4. Add 10 uL mix to well
    5. Run gel for 25 minutes at 250V
    6. Stain for 3 min with 3 uL SYBR gold
    7. Image in gel doc

Results

File:20160829-X2Dilution-Primers-qPCR.png