Daniel:Notebook/ComboLock/2016-6-24: Difference between revisions

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>Djacobse
>Djacobse
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<ol start="2">
<ol start="2">
<li>Latch and Padlock Hybridization</li>
<li>Latch and Padlock Hybridization</li>
<ol start="E">
<ol type="A" start="5">
<li>Heat inactivate enzyme by incubating for 2 minutes at 94C</li>
<li>Heat inactivate enzyme by incubating for 2 minutes at 94C</li>
</ol>
</ol>

Revision as of 15:49, 24 June 2016

Stage 2 Test (Started yesterday)

Back to Calendar

Protocol

  1. Latch and Padlock Hybridization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 2 uL exonuclease I (20 units/μL) and 10 uL exonuclease III (100 units/μL) in 1:1 ratio
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. qPCR
    1. Set up reaction according to table
    2. Sample Lane Batch Cells 2X Kapa SYBR qPCR MM 100 uM Primer2 100 uM Primer4-RC H2O Total Volume (uL)
      Sample 1 A1 B1 2 25 1 1 21 50
      Sample 2 A2 B2 2 25 1 1 21 50
      Sample 3 A3 B3 2 25 1 1 21 50
      Sample 4 A4 B4 2 25 1 1 21 50
      Sample 5 A5 B5 2 25 1 1 21 50
      Sample 6 A6 B6 2 25 1 1 21 50
      Sample 7 A7 B7 2 25 1 1 21 50
      Sample 8 A8 B8 2 25 1 1 21 50
    3. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x20
      7. 72C 2 min
      8. 16C hold