Daniel:Notebook/ComboLock/2017-2-3: Difference between revisions

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>Djacobse
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<li>Add 47 uL master mix to each well</li>
<li>Add 47 uL master mix to each well</li>
<li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section</li>
<li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section</li>
<li>qPCR Cycles</li>
<ol type="a">
<li>95C 3 min</li>
<li>95C 3 sec</li>
<li>55C 30 sec</li>
<li>72C 20 sec</li>
<li>plate read</li>
<li>goto b x24</li>
<li>72C 2 min</li>
<li>16C hold</li>
</ol></ol>
<li>TBE Gel</li>
<ol type="A">
<li>Mix 80 uL TBE, 20 uL 6x loading dye</li>
<li>Aliquot 10 uL per sample/ladder lane onto parafilm</li>
<li>Add 2 uL of sample or ladder to correct drop</li>
<li>Load 10 uL in to well</li>
<li>Run gel for 22 minutes at 250V</li>
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li>
<li>Rinse gel and image in gel doc</li>
</ol></ol>
===Results===
<gallery perrow=2 heights=300px widths=300px>
File:|qPCR Curve
File:|Gel Image
</gallery>
[[Category:ComboLock]] [[Category:20170202]]

Revision as of 17:23, 3 February 2017

Version 2 Oligos Round 2 (Started yesterday)

Back to Calendar

Protocol-Part 4-RCA

  1. Exonuclease Digestion
    1. Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1 hour at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Rolling Circle Amplification
    1. Prepare 8.2X Master mix according to table below
    2. Reagent uL Added Master Mix (8.2X)
      Template 5 NA
      RCA Primer (10 uM) 2.5 20.5
      dNTP (1 mM) 5 41
      10X Buffer 2 16.4
      Phi29 1 0
      BSA (10 mg/mL) 0.4 3.28
      nfH2O 4.1 33.62
      Total 20 131.2
    3. Add 14 uL master mix to each tube
    4. Add sample to new reaction tubes
    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step

Protocol Part 5-qPCR and Gel

  1. qPCR
    1. Make 9.2X qPCR master mix according to following recipe
      1. 193.2 uL nfH2O
      2. 230 uL SYBR Fast
      3. 9.2 uL 10 uM AmpF
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section
    4. 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 80 uL TBE, 20 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

Results