Daniel:Notebook/ComboLock/2017-1-24

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Circularization Test (Started Yesterday)[edit]

Back to Calendar

Protocol-Part 2[edit]

  1. Exonuclease Digestion
    1. Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1.5 hours at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Rolling Circle Amplification
    1. Prepare 7X Master mix according to table below
    2. Add 15 uL master mix to each tube
    3. Add sample to new reaction tubes according to following table
    4. Reagent uL Added Master Mix (8.5X)
      Template 5 NA
      RCA Primer (10 uM) 2.5 21.25
      dNTP (1 mM) 0.8 6.8
      10X Buffer 2 17
      Phi29 1 8.5
      nfH2O 8.7 73.95
      Total 20 127.5
    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step


    Protocol-Part 3[edit]

  3. qPCR
    1. Make qPCR master mix according to following recipes
    2. Master Mix Primer Pair uL Forward Primer uL Reverse Primer uL nfH2O uL 2X SYBR Fast MM uL Total
      MM1 AmpF/AmpR 17.2 None 361.2 430 808.4
      MM2 primer6RC/AmpR.Ind29 9.2 9.2 193.2 230 441.6
    3. Add 47 (MM1) or 48 (MM2) uL master mix to each well
    4. Add 2 uL sample and/or 1 uL appropriate Reverse Primer (1 uM) according to table in following section
    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 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 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 Product Amt Pre/Post RCA Primer Pair Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL) Master Mix
Sample 0A 1pmol Pre AmpF/AmpR A1 21 2 25 1 1 21 50 MM1
Sample 0B 1pmol Pre AmpF/AmpR A2 21 2 25 1 1 21 50 MM1
Sample 2A 10 fmol Pre AmpF/AmpR A3 22 2 25 1 1 21 50 MM1
Sample 2B 10 fmol Pre AmpF/AmpR A4 22 2 25 1 1 21 50 MM1
Sample 4A 100 amol Pre AmpF/AmpR A5 23 2 25 1 1 21 50 MM1
Sample 4B 100 amol Pre AmpF/AmpR A6 23 2 25 1 1 21 50 MM1
Sample 6A 1 amol Pre AmpF/AmpR A7 24 2 25 1 1 21 50 MM1
Sample 6B 1 amol Pre AmpF/AmpR A8 24 2 25 1 1 21 50 MM1
Sample 0AX 1pmol Post AmpF/AmpR H1 25 2 25 1 1 21 50 MM1
Sample 0BX 1pmol Post AmpF/AmpR H2 25 2 25 1 1 21 50 MM1
Sample 2AX 10 fmol Post AmpF/AmpR H3 26 2 25 1 1 21 50 MM1
Sample 2BX 10 fmol Post AmpF/AmpR H4 26 2 25 1 1 21 50 MM1
Sample 4AX 100 amol Post AmpF/AmpR H5 27 2 25 1 1 21 50 MM1
Sample 4BX 100 amol Post AmpF/AmpR H6 27 2 25 1 1 21 50 MM1
Sample 6AX 1 amol Post AmpF/AmpR H7 28 2 25 1 1 21 50 MM1
Sample 6BX 1 amol Post AmpF/AmpR H8 28 2 25 1 1 21 50 MM1
Sample 0A 1pmol Pre primer6RC/AmpR C1 29 2 25 1 1 21 50 MM2
Sample 0B 1pmol Pre primer6RC/AmpR C2 29 2 25 1 1 21 50 MM2
Sample 6A 1 amol Pre primer6RC/AmpR C3 29 2 25 1 1 21 50 MM2
Sample 6B 1 amol Pre primer6RC/AmpR C4 29 2 25 1 1 21 50 MM2
Sample 0AX 1pmol Post primer6RC/AmpR C5 29 2 25 1 1 21 50 MM2
Sample 0BX 1pmol Post primer6RC/AmpR C6 29 2 25 1 1 21 50 MM2
Sample 6AX 1 amol Post primer6RC/AmpR C7 29 2 25 1 1 21 50 MM2
Sample 6BX 1 amol Post primer6RC/AmpR C8 29 2 25 1 1 21 50 MM2
NTC qPCR Primers Only NA AmpF/AmpR F1 30 2 25 1 1 23 50 MM1
NTC qPCR Primers Only NA primer6RC/AmpR F2 30 2 25 1 1 23 50 MM2

Results-Curves and Gel Images[edit]

Results-Cycle Threshold[edit]

Sample Lanes CT Avg CT Diff to PreRCA Amplification CT Diff to 1 amol Amplification to 1 amol
1 pmol PreRCA A1,A2 17.6 NA NA 1.2 2.3
10 fmol PreRCA A3,A4 19.1 NA NA -0.1 0.8
100 amol PreRCA A5,A6 18.9 NA NA -0.2 1.0
1 amol PreRCA A7,A8 18.9 NA NA 0.0 1.0
1 pmol PostRCA H1,H2 11.4 6.25 75.8 6.9 119.8
10 fmol PostRCA H3,H4 16.3 2.78 6.8 2.0 3.9
100 amol PostRCA H5,H6 18.2 0.69 1.6 0.1 1.1
1 amol PostRCA H7,H8 18.3 0.56 1.5 0.0 1.0
1 pmol PreRCA Adapters C1,C2 16.1 NA NA 5.1 33.4
1 amol PreRCA Adapters C3,C4 21.1 NA NA 0.0 1.0
1 pmol PostRCA Adapters C5,C6 11.3 4.8 27.5 9.8 916.5
1 amol PostRCA Adapters C7,C8 20.8 0.4 1.3 0.4 1.3