Daniel:Notebook/ComboLock/2016-10-27

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Revision as of 18:25, 27 October 2016 by >Djacobse (Created page with "=Cell Test 3 (Started Friday 10/21)= Back to Calendar ==Latch PCR== Just testing the PCR of Latch0001...")
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Cell Test 3 (Started Friday 10/21)

Back to Calendar

Latch PCR

Just testing the PCR of Latch0001, which didn't show up at all in the gels from Tuesday. I'm just going to test on the final product, but I'll try to make sure that we get the cells from the bottom of the tubes.

  1. qPCR
    1. Prepare the following master mix
      1. 128.1 uL nfH2O
      2. 6.1 uL primer4
      3. 6.1 uL primer6RC
      4. 152.5 uL Kapa SYBR Fast master mix
    2. Add 48 uL master mix to each well
    3. Add 2 uL sample to correct well by following sample table
    4. Sample Condition Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM p6RC (fwd) Primer 10 uM p4 (rev) Primer H2O Total Volume (uL)
      Sample 1 Exp. Rep 1 A1 2 25 1 1 21 50
      Sample 2 Exp. Rep 2 B1 2 25 1 1 21 50
      Sample 3 No C Probes C1 2 25 1 1 21 50
      Sample 4 No Cells D1 2 25 1 1 21 50
      NTC qPCR primers only E1 0 25 1 1 21 50
      Positve Control Direct Latch0001 (10 uM) F1 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 200 uL TBE, 50 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