Daniel:Notebook/ComboLock/2017-4-3: Difference between revisions

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>Djacobse
>Djacobse
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<li>72C for 2 min</li></ol>
<li>72C for 2 min</li></ol>
</ol>
</ol>
<li>TBE Gel ([[Daniel:Notebook/ComboLock/2017-3-29|Post-Bglii samples]], before Qiaquick column)</li>
 
==Protocol-[[Daniel:Notebook/ComboLock/2017-3-29|Post-Bglii samples]]==
 
<li>TBE Gel</li>
<ol type="A">
<ol type="A">
<li>Mix 80 uL TBE and 20 uL 6X dye</li>
<li>Mix 80 uL TBE and 20 uL 6X dye</li>
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<li>Spin for 1 minute at 14000 rpm</li>
<li>Spin for 1 minute at 14000 rpm</li>
</ol>
</ol>
<li>TBE Gel</li>
 
<li>Bgl Digestion ([[Daniel:Notebook/ComboLock/2017-3-26|Pre-Bglii samples]])</li>
==Protocol-[[Daniel:Notebook/ComboLock/2017-3-26|Pre-Bglii samples]]==
 
<li>Bgl Digestion</li>
<ol type="A">
<ol type="A">
<li>Make the following reactions; '''Do not add Bglii yet'''</li>
<li>Make the following reactions; '''Do not add Bglii yet'''</li>

Revision as of 21:57, 3 April 2017

RCA Test (Started March 24)

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I'll try qPCR on the RCA product again, this time on the NEB 12 hr samples (since I am running low on X samples), but for few cycles and with only the forward primer (which should be RC to the RCA product. This will be fed into Bglii digestion

  1. qPCR-Second strand synthesis
    1. Make the following 13.2X master mix
      1. 13.2 uL AmpF-6.4Sol (10 uM)
      2. 264 uL Kapa SYBR 2X Master Mix
      3. 224.4 uL nfH2O
    2. Aliquot 38 uL master mix into each lane
    3. Add 2 uL sample according to plate layout
    4. File:PlateLayout-20170403-RCATest-CPLP-ssSynthesis.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

    Protocol-Post-Bglii samples

  2. TBE Gel
    1. Mix 80 uL TBE and 20 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample/ladder
    3. Add 2.5 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
  3. Qiaquick Column (Post-Bglii samples)
    1. Add 200 uL (5X) PB buffer to 40 uL sample
    2. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    3. Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through
    4. Dry spin column for 1 minute at 14000 rpm; discard flow through
    5. Let stand with cover open in fume hood for ~5 minutes
    6. Transfer column to a new 1.5 mL eppendorf tube
    7. Add 35 uL nfH2O to column
    8. Let stand 1 minute
    9. Spin for 1 minute at 14000 rpm

    Protocol-Pre-Bglii samples

  4. Bgl Digestion
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol Master Mix (30.2X)
      RCA Rxn 2 0
      Bglii Cutter Primer + (100 uM) 2 60.4
      10X Buffer 3.1 2 60.4
      Bglii 1 0
      nfH2O 13 392.6
      Total 20 513.4
    3. Use the following thermocycler program
      1. 5 min 95C
      2. Ramp to 50C at 0.2C/s
      3. 10 min 50C
      4. 2 hr 37C; When this cycle starts add 1 uL Bglii
      5. Heat kill with 65C for 20 min