Daniel:Notebook/ComboLock/2016-8-23

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Positive Control Amplicon Test 2 (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 20 uL exonuclease I (20 units/μL) and 4 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 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 X2-1X A1 33 2 25 0.5 5 17.5 50
      Sample 2 X2-5X B1 34 2 25 0.5 5 17.5 50
      Sample 3 X2-10X C1 35 2 25 0.5 5 17.5 50
      Sample 4 X2-50X D1 36 2 25 0.5 5 17.5 50
      Sample 5 X3-1X E1 37 2 25 0.5 5 17.5 50
      Sample 6 X3-5X F1 38 2 25 0.5 5 17.5 50
      Sample 7 X3-10X G1 39 2 25 0.5 5 17.5 50
      Sample 8 X3-50X H1 40 2 25 0.5 5 17.5 50
      Sample 9 X2Flip A12 41 2 25 0.5 5 17.5 50
      Sample 10 X3Flip B12 42 2 25 0.5 5 17.5 50
      Sample 9 PCR NTC C12 43 2 25 0.5 5 19.5 50
    3. Master Mix
      1. 277.5 uL KAPA SyberFast MM
      2. 194.25 uL nfH2O
      3. 5.55 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

    Positive Control Amplicon Production

    1. Phosphorylation
      1. In a 0.2 mL tube, add ingredients according to table
      2. Incubate at 37C for 30 min
    2. Ligation
      1. Set up 2 reactions with the following reagents (DO NOT ADD LIGASE YET)
      2. Add amplicons according to following table
      3. Heat reaction to 95C for 5 min
      4. Lower temp to 55C
      5. Add 1 uL Amp Ligase to each reaction without removing from thermocycler; swirl with pipette tip 5 times to mix
      6. Incubate at 55C for 2 hours
      7. Heat to 95 C to denature dsDNA
      8. Purify with ssDNA column
    3. ssDNA Column
      1. Add 100 uL Binding Buffer to the sample; mix well
      2. Transfer to IIC Column and centrifuge at 14000 rpm for 1 minute; SAVE THE FLOW THROUGH
      3. Add 150 uL 100% EtOH to flow through; mix well
      4. Transfer to IC Column and centrifuge at 14000 rpm for 1 minute; discard flow through
      5. Add 400 uL Prep Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
      6. Add 700 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
      7. Add 400 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
      8. Centrifuge empty column at 14000 rpm for 2 minutes
      9. Transfer to empty 1.5mL centrifuge tube (low bind)
      10. Add 30 uL nfH2O and centrifuge at 14000 rpm for 1 minute