Daniel:Notebook/ComboLock/2016-8-19: Difference between revisions

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<gallery perrow=2 heights=300px widths=300px>
<gallery perrow=2 heights=300px widths=300px>
File:20160819-qPCR-PosControlTest.png|qPCR Curve
File:20160819-qPCR-PosControlTest.png|qPCR Curve
File:|Gel Image
File:2016-08-19-PosControl-qPCR.png|Gel Image
</gallery>
</gallery>


[[Category:ComboLock]] [[Category:20160818]]
[[Category:ComboLock]] [[Category:20160818]]

Revision as of 19:52, 19 August 2016

Positive Control Amplicon Test (Started yesterday)

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Protocol

  1. Circularization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 15 uL exonuclease I (20 units/μL) and 3 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. Elution
    1. Add 25 uL prewarmed elution buffer; vortex to suspend
    2. Incubate at RT for 2 minutes
    3. Apply magnet and transfer supernatant to a clean centrifuge tube
    4. Repeat elution with 25 uL fresh elution buffer; add supernatant to first elution
  4. qPCR
    1. Set up reaction according to table
    2. Sample Name Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM AmpF 6.4 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1 Amp1 Test A1 10 2 25 0.5 5 17.5 50
      Sample 2 Amp3 Test B1 11 2 25 0.5 5 17.5 50
      Sample 3 Latch X2 Specificity C1 12 2 25 0.5 5 17.5 50
      Sample 4 Latch X3 Specificity D1 13 2 25 0.5 5 17.5 50
      Sample 5 No Template E1 14 2 25 0.5 5 17.5 50
      Sample 6 No Latch F1 15 2 25 0.5 5 17.5 50
      Sample 7 No Padlock G1 16 2 25 0.5 5 17.5 50
      Sample 8 PCAmp2 Only H1 17 2 25 0.5 5 17.5 50
      Sample 9 PCR NTC A12 18 0 25 0.5 5 19.5 50
    3. Master Mix
      1. 227.5 uL KAPA SyberFast MM
      2. 159.25 uL nfH2O
      3. 4.55 uL AmpF 6.4
    4. Add 43 uL Master Mix to each well
    5. 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

    Results