Daniel:Notebook/ComboLock/2016-6-24

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Revision as of 18:10, 24 June 2016 by >Djacobse (→‎Protocol)
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Stage 2 Test (Started yesterday)

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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 *this is actually PCR since the eppendorf thermocycler was down and the other was in use
    1. Set up reaction according to table
    2. Sample Lane Batch AmpR Index Cells 2X Kapa SYBR qPCR MM 100 uM AmpF 6.4 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1 A1 B1 1 2 25 0.5 5 17.5 50
      Sample 2 A2 B2 2 2 25 0.5 5 17.5 50
      Sample 3 A3 B3 3 2 25 0.5 5 17.5 50
      Sample 4 A4 B4 4 2 25 0.5 5 17.5 50
      Sample 5 A5 B5 5 2 25 0.5 5 17.5 50
      Sample 6 A6 B6 6 2 25 0.5 5 17.5 50
      Sample 7 A7 B7 7 2 25 0.5 5 17.5 50
      Sample 8 A8 B8 8 2 25 0.5 5 17.5 50
    3. Master Mix
      1. 200 uL KAPA SyberFast MM
      2. 140 uL nfH2O
      3. 4 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
  4. PAGE gel
    1. Set up lanes according to table
    2. Sample Lane uL TBE Buffer uL loading dye uL sample
      Ladder 2 4 2 2
      Batch1 3 4 2 2
      Batch2 4 4 2 2
      Batch3 5 4 2 2
      Batch4 6 4 2 2
      Batch5 7 4 2 2
      Batch6 8 4 2 2
      Batch7 9 4 2 2
      Batch8 10 4 2 2
    3. Run gel for 25 minutes at 220V