Daniel:Notebook/ComboLock/2016-10-19

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Revision as of 18:39, 19 October 2016 by >Djacobse (Created page with "=Cell Trial 2 (Started Yesterday)= Back to Calendar ==Protocol== <ol start="3"> <li>Circularization</...")
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Cell Trial 2 (Started Yesterday)

Back to Calendar

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 7.5 uL exonuclease I (20 units/μL) and 1.5 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. Prepare the following master mix
      1. 89.25 uL nfH2O
      2. 2.55 uL 100 uM AmpF6.4Sol
      3. 127.5 uL Kapa SYBR Fast MM
    2. Add 43 uL master mix to each well
    3. Add sample and AmpR indexes according to sample table below
    4. Sample Condition Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM AmpF6.4Sol 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1 Exp. Rep 1 A1 20 2 25 0.5 5 17.5 50
      Sample 2 Exp. Rep 2 B1 20 2 25 0.5 5 17.5 50
      Sample 3 No C Probes C1 21 2 25 0.5 5 17.5 50
      Sample 4 No Cells D1 22 2 25 0.5 5 17.5 50
      NTC No qPCR sample E1 23 0 25 0.5 5 19.5 50
    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 x24
      7. 72C 2 min
      8. 16C hold
  4. TBE Gel
    1. Mix 56 uL TBE, 14 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