Daniel:Notebook/ComboLock/2016-11-1

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Circularization Optimization Testing (Started Yesterday)[edit]

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Sample Matrix[edit]

Sample Polymerase Incubation Time RCA Y/N
Sample 1 (AB) Klentaq ON Y
Sample 2 (AB) Klentaq ON N
Sample 3 (AB) Klentaq 2 hr Y
Sample 4 (AB) Klentaq 2 hr N
Sample 5 (AB) Phusion ON Y
Sample 6 (AB) Phusion ON N
Sample 7 (AB) Phusion 2 hr Y
Sample 8 (AB) Phusion 2 hr N

Protocol[edit]

  1. Circularization (all samples)
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 30 uL exonuclease I (20 units/μL) and 6 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. Rolling Circle Amplification (Samples 1,3,5,7; Hold samples 2,4,6,8 at 4C)
    1. Prepare 8.1X Master mix according to table below
    2. Add sample to new reaction tubes according to following table
    3. Reagent uL Added Master Mix (8.1X)
      Template 5 NA
      RCA Primer (100 uM) 2.5 20.25
      dNTP (1 mM) 0.8 6.48
      10X Buffer 2 16.2
      Phi29 1 8.1
      nfH2O 8.7 70.47
      Total 20 121.5
    4. Incubate at 37C for 3 hours
    5. Incubate at 65C for 10 minutes
    6. Hold at 10C
  4. qPCR
    1. Set up reaction according to table below
    2. Make master mixes according to following recipes
      1. 535.5 uL nfH2O
      2. 637.5 uL SYBR Fast
      3. 25.5 uL 10 uM AmpF
    3. Add 47 uL master mix to each well
    4. 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
  5. TBE Gel
    1. Mix 240 uL TBE, 60 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

qPCR Sample Table[edit]

Sample Condition RCA? Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
Sample 1A K-ON-Y N A1 20 2 25 1 1 21 50
Sample 1B K-ON-Y N A2 20 2 25 1 1 21 50
Sample 2A K-ON-N N A3 21 2 25 1 1 21 50
Sample 2B K-ON-N N A4 21 2 25 1 1 21 50
Sample 3A K-2H-Y N A5 22 2 25 1 1 21 50
Sample 3B K-2H-Y N A6 22 2 25 1 1 21 50
Sample 4A K-2H-N N A7 23 2 25 1 1 21 50
Sample 4B K-2H-N N A8 23 2 25 1 1 21 50
Sample 5A P-ON-Y N F1 24 2 25 1 1 21 50
Sample 5B P-ON-Y N F2 24 2 25 1 1 21 50
Sample 6A P-ON-N N F3 25 2 25 1 1 21 50
Sample 6B P-ON-N N F4 25 2 25 1 1 21 50
Sample 7A P-2H-Y N F5 26 2 25 1 1 21 50
Sample 7B P-2H-Y N F6 26 2 25 1 1 21 50
Sample 8A P-2H-N N F7 27 2 25 1 1 21 50
Sample 8B P-2H-N N F8 27 2 25 1 1 21 50
Sample 1A K-ON-Y Y H1 28 2 25 1 1 21 50
Sample 1B K-ON-Y Y H2 28 2 25 1 1 21 50
Sample 3A K-2H-Y Y H3 29 2 25 1 1 21 50
Sample 3B K-2H-Y Y H4 29 2 25 1 1 21 50
Sample 5A P-ON-Y Y H5 30 2 25 1 1 21 50
Sample 5B P-ON-Y Y H6 30 2 25 1 1 21 50
Sample 7A P-2H-Y Y H7 31 2 25 1 1 21 50
Sample 7B P-2H-Y Y H8 31 2 25 1 1 21 50
NTC NA N C1 33 2 25 1 1 21 50

Results[edit]