Daniel:Notebook/ComboLock/2016-12-15

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Revision as of 18:36, 15 December 2016 by >Djacobse (→‎Results)
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Mirror Probe Test (Started Tuesday)

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RCA on Beads/Supernatant Fractions

  1. qPCR
    1. Make master mix according to following recipe
      1. 298.2 uL nfH2O
      2. 355 uL SYBR Fast
      3. 14.2 uL 10 uM AmpF
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section
    4. Sample Condition Fraction Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 1A Sample Supernatant A1 20 2 25 1 1 21 50
      Sample 1B Sample Supernatant A2 20 2 25 1 1 21 50
      Sample 2A No Template Supernatant A3 21 2 25 1 1 21 50
      Sample 2B No Template Supernatant A4 21 2 25 1 1 21 50
      Sample 3A No C Probe Supernatant A5 22 2 25 1 1 21 50
      Sample 3B No C Probe Supernatant A6 22 2 25 1 1 21 50
      Sample 1A Sample Beads H1 23 2 25 1 1 21 50
      Sample 1B Sample Beads H2 23 2 25 1 1 21 50
      Sample 2A No Template Beads H3 24 2 25 1 1 21 50
      Sample 2B No Template Beads H4 24 2 25 1 1 21 50
      Sample 3A No C Probe Beads H5 25 2 25 1 1 21 50
      Sample 3B No C Probe Beads H6 25 2 25 1 1 21 50
      NTC qPCR Primers Only NA A7 26 2 25 1 1 23 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 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

Results

C Probe and Product Detection