Daniel:Notebook/ComboLock/2017-1-18

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Revision as of 00:38, 19 January 2017 by >Djacobse (→‎Product Check)
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Version 3 Oligos (Started Thursday, Jan 13)

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Product Check

Searching for the bridge product (or circular product) using the primer24/primer12 combination.

  1. qPCR
    1. Make 5.2X qPCR master mix according to following recipe
      1. 109.2 uL nfH2O
      2. 130 uL SYBR Fast
      3. 5.2 uL 10 uM primer24RC
      4. 5.2 uL 10 uM primer24RC
    2. Add 48 uL master mix to each well
    3. Add 2 uL sample according to table
    4. Sample Condition Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 1A Sample A1 2 25 1 1 21 50
      Sample 1B Sample A2 2 25 1 1 21 50
      Sample 4A No Lock Phosphate A3 2 25 1 1 21 50
      Sample 4B No Lock Phosphate A4 2 25 1 1 21 50
      NTC qPCR Primers Only A5 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 64 uL TBE, 16 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 230V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results

RCA Repeat

Gonna try RCA again, since the adapter testing above showed product with at least the adapter regions. That means either the bridge is present or the circular product itself. So repeating RCA should increase the concentration of product, if available.

  • Rolling Circle Amplification
    1. Prepare 9X Master mix according to table below
    2. Add 15 uL master mix to each tube
    3. Add sample to new reaction tubes according to following table
    4. Reagent uL Added Master Mix (8.5X)
      Template 5 NA
      RCA Primer (10 uM) 2.5 21.25
      dNTP (1 mM) 0.8 6.8
      10X Buffer 2 17
      Phi29 1 8.5
      nfH2O 8.7 73.95
      Total 20 127.5