Daniel:Notebook/ComboLock/2017-3-16: Difference between revisions

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>Djacobse
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<li>Exonuclease Digestion</li>
<li>Exonuclease Digestion</li>
<ol type="A">
<ol type="A">
<li>Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)</li>
<li>Mix 25 uL Exo I (20U/uL) and 5 uL ExoIII (100U/uL)</li>
<li>Add 2 uL to each sample, mix by swirling pipette tip</li>
<li>Add 2 uL to each sample, mix by swirling pipette tip</li>
<li>Incubate for 1.5 hours at 37C</li>
<li>Incubate for 1.5 hours at 37C</li>

Revision as of 19:44, 16 March 2017

Circularization Comparison (T4 Poly/Ligase)

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Protocol-Part 4

  1. Exonuclease Digestion
    1. Mix 25 uL Exo I (20U/uL) and 5 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1.5 hours at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. qPCR
    1. Make qPCR master mixes according to following recipe
    2. Reagent Single Rxn MMProd (15.2X) MM+ (C1, 4.2X)
      Primer Pair (fwd/rev) NA primer12*/AmpR-Ind21 primer24/primer4*
      Forward Primer (10 uM) 1 15.2 4.2
      Reverse Primer (10 uM) 1 15.2 4.2
      nfH2O 21 319.2 88.2
      2X Kapa SYBR Fast 25 380 105
      Total 48 729.6 201.6
    3. Add 48 uL master mix to each well
    4. Add 2 uL sample according to plate layout
    5. File:PlateLayout-20170316-T4Circ-PreRCATest.png
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold
  3. Rolling Circle Amplification
    1. Samples will be aliquoted under following rules