Daniel:Notebook/ComboLock/2017-4-5

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Revision as of 23:22, 5 April 2017 by >Djacobse (→‎Results)
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RCA Test (Started March 24)

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Second Strand Synthesis part II

Since the second strand synthesis from Monday worked well, I'm going to repeat the experiment but using the NEB 3 hr and 6 hr samples (X and P, respectively). I won't bother with the Bgl digested ones as I'll just digest after.

  1. qPCR-Second strand synthesis
    1. Make the following 14.2X master mix
      1. 14.2 uL AmpF-6.4Sol (10 uM)
      2. 284 uL Kapa SYBR 2X Master Mix
      3. 241.4 uL nfH2O
    2. Aliquot 38 uL master mix into each lane
    3. Add 2 uL sample according to plate layout
    4. File:PlateLayout-qPCR-20170405-RCATest-CPLP-ssSynthesis2.png
    5. Run the following protocol
      1. 95C for 2 min
      2. 95C for 5 sec
      3. 55C for 30 sec
      4. 72C for 45 sec
      5. Goto (b) x20
      6. 72C for 2 min
  2. Bgl Digestion
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol Master Mix (12.2X)
      RCA Rxn 8 0
      10X Buffer 3.1 2 24.4
      Bglii 1 0
      nfH2O 9 111.6
      Total 20 136
    3. Aliquot 11 uL master mix into each sample
    4. Add appropriate sample and 1 uL Bglii to each
    5. Incubate 1 hr 37C
    6. Heat kill with 65C for 20 min
  3. TBE Gel-PreBgl
    1. Mix 96 uL 10X TBE and 24 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample
    3. Add 2 uL reaction or 1.5 uL ladder to appropriate aliquot
    4. Add 10 uL mix to gel lanes
    5. File:GelLanes-20170405-RCATest-CPLP-PostqPCR-PreBgl.png
    6. Run gel for 24 minutes at 230V
    7. Stain with 2 uL SYBR gold for 3 minutes
    8. Rinse and image in gel doc
  4. TBE Gel-PostBgl
    1. Mix 120 uL 10X TBE and 40 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot
    3. Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot
    4. Add 10 uL mix to gel lanes
    5. Run gel for 24 minutes at 230V
    6. Stain with 2 uL SYBR gold for 3 minutes
    7. Rinse and image in gel doc

Results

<gallery perrow=3 heights=300px widths=300px> File:20170405-qPCR-RCATest-CPLP-ssSynthesis2.png|qPCR curves File:2017-04-05_-RCATest-CPLP-ssSynthesis2-PreBgl.png|Gel image-pre Bgl digestion File:|Gel image-post Bgl digestion