Daniel:Notebook/ComboLock/2016-11-30

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Extended Latch Test (Started Nov 29)[edit]

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Sample Matrix[edit]

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No Phosphate Ligation
Sample 4 (AB) No C Probe

Protocol[edit]

  1. Exonuclease Digestion
    1. Combine 15 uL of Exonuclease I (20U/uL) and 3 uL Exonuclease III (100U/uL)
    2. Add 2 uL of mix to each reaction
    3. Incubate for 30 minutes to 2 hours at 37C
    4. Heat kill by incubating for 5 minutes at 94C
  2. Rolling Circle Amplification
    1. Prepare 9X master mix according to table below
    2. Reagent Stock Concentration Final Conc/Amt uL Added in 20 uL Rxn Master Mix (9X)
      Template NA NA 5 45
      RCA Primer (RCA-CL-LinkerLRC-v01) 10 uM 2 uM 4 36
      dNTP 1 mM 50 uM 1 9
      10X Buffer 10X 1X 2 18
      Phi29 (NEB) 10 U/uL 10 U 1 9
      nfH2O NA NA 7 63
      Total     20 180
    3. Add 15 uL master mix to each tube
    4. Add 5 uL sample to correct reaction tubes
    5. Incubate at 37C for 3 hours
    6. Heat kill by incubating at 65C for 10 minutes
  3. qPCR
    1. Make master mix according to following recipe
      1. 359.1 uL nfH2O
      2. 427.5 uL SYBR Fast
      3. 17.1 uL 10 uM AmpF
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table
    4. 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 Sample Y A1 20 2 25 1 1 21 50
      Sample 1B Sample Y A2 20 2 25 1 1 21 50
      Sample 2A No Template Y A3 21 2 25 1 1 21 50
      Sample 2B No Template Y A4 21 2 25 1 1 21 50
      Sample 3A No Phosphate Y A5 22 2 25 1 1 21 50
      Sample 3B No Phosphate Y A6 22 2 25 1 1 21 50
      Sample 4A No C Probe Y A7 23 2 25 1 1 21 50
      Sample 4B No C Probe Y A8 23 2 25 1 1 21 50
      Sample 1A Sample N D1 24 2 25 1 1 21 50
      Sample 1B Sample N D2 24 2 25 1 1 21 50
      Sample 2A No Template N D3 25 2 25 1 1 21 50
      Sample 2B No Template N D4 25 2 25 1 1 21 50
      Sample 3A No Phosphate N D5 26 2 25 1 1 21 50
      Sample 3B No Phosphate N D6 26 2 25 1 1 21 50
      Sample 4A No C Probe N D7 27 2 25 1 1 21 50
      Sample 4B No C Probe N D8 27 2 25 1 1 21 50
      NTC qPCR Primers Only NA H1 28 2 25 1 1 23 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 200 uL TBE, 50 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[edit]

You can see that the pre and post-RCA reactions amplify at the same rate and are even similar in their gel smears. Tomorrow I will do some QC testing and try to find where the problems are.