Daniel:Notebook/ComboLock/2016-12-1

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Extended Latch Test (Started Nov 29)

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Sample Matrix

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No phosphate activation
Sample 4 (AB) No C Probe

Beads smear test

One thing I have been doing in the last few weeks is being careful to agitate the beads and get a few into the RCA reaction. I've been doing this in case the circular product is in some way still attached to the beads. However, it could explain why there are smears in both products. I'm going to test a few scenarios to examine this hypothesis.

  • Gel run of unagitated (not containing beads) and agitated (containing beads) media
  • Repeat RCA using unagitated supernatant from the exonuclease reaction

Samples List

Lane Sample Condition
2 25bp Ladder NA
3 Sample A Supernatant
4 Sample B Supernatant
5 Sample A Beads+
6 Sample B Beads+
7 No Template A Supernatant
8 No Template B Supernatant
9 No Template A Beads+
10 No Template B Beads+


  1. TBE Gel
    1. Mix 80 uL TBE, 20 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 25 minutes at 225V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

File:2016-12-01-ExtLatch-QC1.png

Doesn't really show anything, the concentration of both is too low.

No Beads RCA

  1. 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 NA
      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     20 135
    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
  2. 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 A1 24 2 25 1 1 21 50
      Sample 1B Sample N A2 24 2 25 1 1 21 50
      Sample 2A No Template N A3 25 2 25 1 1 21 50
      Sample 2B No Template N A4 25 2 25 1 1 21 50
      Sample 3A No Phosphate N A5 26 2 25 1 1 21 50
      Sample 3B No Phosphate N A6 26 2 25 1 1 21 50
      Sample 4A No C Probe N A7 27 2 25 1 1 21 50
      Sample 4B No C Probe N A8 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
  3. 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

Adapter Amplification

  1. 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 pimer24RC
      4. 17.1 uL 10 uM pimer12
    2. Add 48 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 Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 1A Sample A1 2 25 1 1 21 50
      Sample 1B Sample A2 2 25 1 1 21 50
      Sample 2A No Template A3 2 25 1 1 21 50
      Sample 2B No Template A4 2 25 1 1 21 50
      Sample 3A No Phosphate A5 2 25 1 1 21 50
      Sample 3B No Phosphate A6 2 25 1 1 21 50
      Sample 4A No C Probe A7 2 25 1 1 21 50
      Sample 4B No C Probe A8 2 25 1 1 21 50
      Sample 1A Sample D1 2 25 1 1 21 50
      Sample 1B Sample D2 2 25 1 1 21 50
      Sample 2A No Template D3 2 25 1 1 21 50
      Sample 2B No Template D4 2 25 1 1 21 50
      Sample 3A No Phosphate D5 2 25 1 1 21 50
      Sample 3B No Phosphate D6 2 25 1 1 21 50
      Sample 4A No C Probe D7 2 25 1 1 21 50
      Sample 4B No C Probe D8 2 25 1 1 21 50
      NTC qPCR Primers Only H1 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
  2. TBE Gel
    1. Mix 176 uL TBE, 44 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