Daniel:Notebook/ComboLock/2016-10-25

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Cell Test 3 (Started Friday 10/21)[edit]

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Latch-Padlock Detection qPCR[edit]

  1. qPCR
    1. Prepare the following master mixes according to table
    2. Master Mix Forward/Reverse Target Forward Primer 10 uM (uL) Reverse Primer10 uM (uL) nfH2O (uL) Kapa SYBR Fast MM (uL)
      MM1 p24RC/p12 Padlock0201 10.1 10.1 212.1 252.5
      MM2 p4/p6RC Latch0001 10.1 10.1 212.1 252.5
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample to correct well by following sample table in next section
    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

qPCR Sample Table[edit]

Sample Condition Time Point Lane Latch/Padlock Target Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL) Master Mix
Sample 1 Exp. Rep 1 T3 A1 Padlock 2 25 1 1 21 50 MM1
Sample 2 Exp. Rep 2 T3 B1 Padlock 2 25 1 1 21 50 MM1
Sample 3 No C Probes T3 C1 Padlock 2 25 1 1 21 50 MM1
Sample 4 No Cells T3 D1 Padlock 2 25 1 1 21 50 MM1
Sample 1 Exp. Rep 1 T3 E1 Latch 2 25 1 1 21 50 MM2
Sample 2 Exp. Rep 2 T3 F1 Latch 2 25 1 1 21 50 MM2
Sample 3 No C Probes T3 G1 Latch 2 25 1 1 21 50 MM2
Sample 4 No Cells T3 H1 Latch 2 25 1 1 21 50 MM2
Sample 1 Exp. Rep 1 T4 A2 Padlock 2 25 1 1 21 50 MM1
Sample 2 Exp. Rep 2 T4 B2 Padlock 2 25 1 1 21 50 MM1
Sample 3 No C Probes T4 C2 Padlock 2 25 1 1 21 50 MM1
Sample 4 No Cells T4 D2 Padlock 2 25 1 1 21 50 MM1
Sample 1 Exp. Rep 1 T4 E2 Latch 2 25 1 1 21 50 MM2
Sample 2 Exp. Rep 2 T4 F2 Latch 2 25 1 1 21 50 MM2
Sample 3 No C Probes T4 G2 Latch 2 25 1 1 21 50 MM2
Sample 4 No Cells T4 H2 Latch 2 25 1 1 21 50 MM2
NTC No qPCR sample NA A12 Padlock 0 25 1 1 23 50 MM1
NTC No qPCR sample NA B12 Latch 0 25 1 1 23 50 MM2
PC-Padlock No qPCR sample NA C12 Padlock 2 25 1 1 21 50 MM1
PC-Latch No qPCR sample NA D12 Latch 2 25 1 1 21 50 MM2

Results[edit]