Daniel:Notebook/ComboLock/2016-10-24

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

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I'm going to try some more qPCR runs in order to delve into this deeper. For one, I'm going to repeat the qPCR from Saturday for time points T1 and T2, since the results are a little strange (I saw nothing in T1 and T2). Second, I'm going to test the adapter based amplifications for latch padlock for several time points, including the last product.

qPCR Repeats[edit]

  1. qPCR
    1. Prepare the following master mixes according to table
    2. Master Mix Forward/Reverse Forward Primer (uL) Reverse Primer (uL) nfH2O (uL) Kapa SYBR Fast MM (uL)
      MM1 p24RC/p4 0.9 0.9 207.5 227.5
      MM2 p6RC/p12 0.9 0.9 207.5 227.5
    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 56 uL TBE, 14 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 AmpR-F Combo Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM Forward Primer 100 uM Reverse Primer H2O Total Volume (uL) Master Mix
Sample 1 Exp. Rep 1 T1 A1 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 2 Exp. Rep 2 T1 B1 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 3 No C Probes T1 C1 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 4 No Cells T1 D1 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 1 Exp. Rep 1 T1 E1 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 2 Exp. Rep 2 T1 F1 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 3 No C Probes T1 G1 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 4 No Cells T1 H1 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 1 Exp. Rep 1 T2 A3 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 2 Exp. Rep 2 T2 B3 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 3 No C Probes T2 C3 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 4 No Cells T2 D3 p24RC/p4 2 25 0.1 0.1 22.8 50 MM1
Sample 1 Exp. Rep 1 T2 E3 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 2 Exp. Rep 2 T2 F3 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 3 No C Probes T2 G3 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
Sample 4 No Cells T2 H3 p6RC/p12 2 25 0.1 0.1 22.8 50 MM2
NTC No qPCR sample NA A12 p24RC/p4 0 25 0.1 0.1 24.8 50 MM1
NTC No qPCR sample NA B12 p6RC/p12 0 25 0.1 0.1 24.8 50 MM2

Results[edit]

Additional Analysis[edit]

I can test the circular product existence by replacing AmpF with primer12, as before.

  1. qPCR
    1. Prepare the following master mix
      1. 107.1 uL nfH2O
      2. 5.1 uL 10 uM primer 12
      3. 127.5 uL Kapa SYBR Fast
    2. Add 43 uL master mix to each well
    3. Add 2 uL sample and 5 uL correct AmpR Index (10 uM) to correct well by following sample table
    4. Sample Condition Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM primer 12 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1 Exp. Rep 1 A1 20 2 25 1 1 21 50
      Sample 2 Exp. Rep 2 B1 20 2 25 1 1 21 50
      Sample 3 No C Probes C1 21 2 25 1 1 21 50
      Sample 4 No Cells D1 22 2 25 1 1 21 50
      NTC No qPCR sample E1 23 0 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

    Results[edit]