Daniel:Notebook/ComboLock/2017-2-28

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RCA + Circularization Test (Started Yesterday)[edit]

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Protocol-Part 3[edit]

  1. ssQubit
    1. Mix together 796 uL Qubit ssDNA buffer and 4 uL dye (200:1 ratio)
    2. Add 190 uL mix and 10 uL S1 or S2 to standard tubes
    3. Add 199 uL mix and 1 uL sample to sample tubes
    4. Incubate 5 minutes in the dark
    5. Measure in Qubit; use new run and calibrate with standards
    6. Results: Both too low to mean anything.
  2. Column Purification-Qiaquick
    1. Add 250 uL (5X) PB (binding buffer) to sample
    2. Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
    3. Add 750 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through;
    4. Repeat wash step;
    5. Dry spin column for 1 minute at 14000 rpm; discard flow through
    6. Let stand with cover open in fume hood for ~5 minutes
    7. Transfer column to a new 1.5 mL eppendorf tube
    8. Elute with 40 uL EB
    9. Let stand 1 minute
    10. Spin for 1 minute at 14000 rpm
  3. Rolling Circle Amplification
    1. Prepare master mixes with following enzyme layout following the table in this section
    2. Samples Condition
      Sample X (1-2,ABC) NEB
      Sample Y (1-2,ABC) Thermo Fisher
      Sample Z (1-2,ABC) Epicenter
      A Samples (1-2, XYZ) Normal Primer
      B Samples (1-2, XYZ) Phosphorothiorate Primer
      C Samples (1-2, XYZ) Column Purified Product
    3. Add 11.5 uL master mix to each tube
    4. Add 2.5 uL LLRC-01 (10 uM) primer to A and C samples; add 2.5 uL LLRC* (10 uM) primer to B samples
    5. Add 5 uL sample to new reaction tubes according to following table
    6. Reagent Single Rxn Vol MM-X (NEB) uL MM-Y (TF) uL MM-Z (Epi) uL
      Template 5 0 0 0
      RCA Primer (10 uM) 2.5 15.25 15.25 15.25
      dNTP (1 mM) 5 30.5 30.5 30.5
      10X Buffer 2 12.2 12.2 12.2
      Phi29 1 0 0 0
      BSA (10 mg/mL) 0.4 2.44 0 0
      nfH2O 4.1 25.01 27.45 27.45
      Total 20 85.4 85.4 85.4
    7. Incubate for 5 minutes at 75C to denature sticky strands
    8. Lower temperature and incubate for 30 minutes at 55C
    9. Lower temperature to 30C; allow at least 1 minute of 30C incubation
    10. Add 1 uL correct Phi29 polymerase and incubate 3 hours at 30C
    11. Heat kill enzyme with 10 minutes at 65C

Protocol-Part 4[edit]

  1. qPCR
    1. Make 25.2X qPCR master mix according to following recipe
      1. 529.2 uL nfH2O
      2. 630 uL SYBR Fast
      3. 25.2 uL 10 uM AmpF
      4. 25.2 uL 10 uM AmpR-Ind20
    2. Add 48 uL master mix to each well
    3. Add 2 uL sample according to PCR Plate Layout
    4. File:PlateLayout-20170228-RCATest.png
    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 x40
      7. 72C 2 min
      8. 16C hold
  2. 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 23 minutes at 230V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results[edit]