Daniel:Notebook/ComboLock/2016-11-8

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Cell Test with RCA (Started Yesterday)[edit]

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Protocol[edit]

  1. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 15 uL exonuclease I (20 units/μL) and 3 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
  2. Rolling Circle Amplification
    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 A (2.2X) Master Mix B (2.2X) Master Mix C (8.2X)
      Template 5 11 11 NA
      RCA Primer (100 uM) 2.5 5.5 5.5 20.5
      RCA Primer Name NA RCA-CL-v01 Chris' Primer RCA-CL-v01
      dNTP (1 mM) 0.8 1.76 1.76 6.56
      10X Buffer 2 4.4 4.4 16.4
      Phi29 1 2.2 2.2 8.2
      Phi29 Company NA NEB NEB Epicentre
      nfH2O 8.7 19.14 19.14 71.34
      Total 20   44 123
    4. Incubate at 37C for 3 hours
    5. Heat kill enzyme by incubating at 65C for 10 minutes
  3. qPCR
    1. Make master mix according to following recipe
      1. 291.1 uL nfH2O
      2. 227.5 uL SYBR Fast
      3. 9.1 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 Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 1AC Experiment A1 20 2 25 1 1 21 50
      Sample 1BC Experiment A2 20 2 25 1 1 21 50
      Sample 2AC Experiment A3 21 2 25 1 1 21 50
      Sample 2BC Experiment A4 21 2 25 1 1 21 50
      Sample 3AC No C Probes A5 22 2 25 1 1 21 50
      Sample 3BC No C Probes A6 22 2 25 1 1 21 50
      Sample 4AC No Cells A7 23 2 25 1 1 21 50
      Sample 4BC No Cells A8 23 2 25 1 1 21 50
      Sample 1AA Exp-Epicentre H1 24 2 25 1 1 21 50
      Sample 1BA Exp-Epicentre H2 24 2 25 1 1 21 50
      Sample 2AB Exp-Primer2 H3 25 2 25 1 1 21 50
      Sample 2BB Exp-Primer2 H4 25 2 25 1 1 21 50
      NTC   H5 26 0 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 120 uL TBE, 30 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]

Controls Test[edit]

Testing the control samples that I have. I have used several positive controls and received no results when I should. So I'm testing the various control samples to see what comes out of it.

  1. qPCR
    1. Make master mix according to following recipes
    2. Master Mix Primer Pair uL Forward Primer 10 uM) uL Reverse Primer (10 uM) uL 2X Kapa SYBR Fast Master Mix uL nfH2O
      MM1 (4.1X) p24/p4RC 4.1 4.1 102.5 86.1
      MM2 (4.1X) p6/p12RC 4.1 4.1 102.5 86.1
      MM3 (3.1X) p24/p12RC 3.1 3.1 77.5 65.1
      MM4 (3.1X) p6/p4RC 3.1 3.1 77.5 65.1
    3. Add 47 uL master mix to each well
    4. Add 2 uL sample 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 x35
      7. 72C 2 min
      8. 16C hold
  2. TBE Gel
    1. Mix 120 uL TBE, 30 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]

qPCR Sample Table[edit]

Sample Primers Master Mix Lane Expected Product Size (bp) Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
BSA-PA p24/p4RC MM1 A1 44 2 25 1 1 21 50
BSA-PB p6/p12RC MM2 A2 44 2 25 1 1 21 50
BSA-PA p24/p12RC MM3 A3 0 2 25 1 1 21 50
BSA-PB p6/p4RC MM4 A4 0 2 25 1 1 21 50
BSA Sample p24/p4RC MM1 A5 44 2 25 1 1 21 50
BSA Sample p6/p12RC MM2 A6 44 2 25 1 1 21 50
BSA Sample p24/p12RC MM3 A7 0 2 25 1 1 21 50
BSA Sample p6/p4RC MM4 A8 0 2 25 1 1 21 50
PCC-C1 p24/p4RC MM1 H1 44 2 25 1 1 21 50
PCC-C1 p6/p12RC MM2 H2 0 2 25 1 1 21 50
NTC p24/p4RC MM1 H3 0 2 25 1 1 21 50
NTC p24/p12RC MM2 H4 0 2 25 1 1 21 50
NTC p6/p12RC MM3 H5 0 2 25 1 1 21 50
NTC p6/p4RC MM4 H6 0 2 25 1 1 21 50