Daniel:Notebook/ComboLock/2017-3-26

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RCA Test (Started March 24)[edit]

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Linear Product Production-Bglii (Started Yesterday)[edit]

  1. TBE gel
    1. Mix 64 uL TBE and 16 uL 6X dye
    2. Aliquot 10 uL on to parafilm per sample/ladder
    3. Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot
    4. File:GelLanes-20170325-LinearProduct.png
    5. Add 10 uL mix to gel lanes
    6. Run gel for 24 minutes at 230V
    7. Stain with 2 uL SYBR gold for 3 minutes
    8. Rinse and image in gel doc

File:2017-03-26-LinearProductProduction-Bglii.png

That's the right one. However, there is a ton of primer (bottom of the gel), so I'll run a ssDNA column to fix.

  1. ssDNA column
    1. Add 50 uL Binding Buffer to the sample; mix well
    2. Transfer to IIC Column and centrifuge at 14000 rpm for 1 minute; SAVE THE FLOW THROUGH
    3. Add 70 uL 100% EtOH to flow through; mix well
    4. Transfer to IC Column and centrifuge at 14000 rpm for 1 minute; discard flow through
    5. Add 400 uL Prep Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
    6. Add 700 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
    7. Add 400 uL Wash Buffer and centrifuge at 14000 rpm for 1 minute; discard flow through
    8. Centrifuge empty column at 14000 rpm for 2 minutes
    9. Transfer to empty 1.5mL centrifuge tube (low bind)
    10. Add 20 uL nfH2O and centrifuge at 14000 rpm for 1 minute

    RCA Protocol[edit]

    1. Rolling Circle Amplification
      1. Prepare master mix according to table below
      2. Reagent Single Rxn (uL) Master Mix X+ (9.2X) Master Mix X- (9.2X) Master Mix Y+ (3.2X) Master Mix Y- (3.2X) Master Mix Z+ (3.2X) Master Mix Z- (3.2X)
        Enzyme NA NEB NEB Thermo Thermo Epi (1:10) Epi (1:10)
        Circular Product (1X) 2 18.4 0 6.4 0 6.4 0
        Linear Product (1X) 2 0 18.4 0 6.4 0 6.4
        RCA Primer (100 uM) 2.5 23 23 8 8 8 8
        dNTP (1 mM) 5 46 46 16 16 16 16
        10X Buffer 2 18.4 18.4 6.4 6.4 6.4 6.4
        Phi29 1 0 0 0 0 0 0
        BSA (10 mg/mL) 0.4 3.68 3.68 0 0 0 0
        nfH2O 8.1 65.32 65.32 24 24 24 24
        Total 20 117.8 117.8 59.52 59.52 59.52 59.52
      3. Add 16.5 uL master mix to each tube
        1. Samples "A-C" get + master mixes; samples "D-F" get - master mixes
      4. Add 2.5 uL 10 uM RCA primer and to new reaction tubes according to following rules
        1. "A,D" samples get LLRC
        2. "B,E" samples get LLRC-v03 (Mono-phosphorothiorate)
        3. "C,F" samples get LLRC-v04 (Tri-phosphorothiorate)
      5. Heat reactions up to 95C for 5 min
      6. Cool to 55C and incubate for 15 minutes
      7. Cool to 30C and add enzyme
      8. Incubate at 30C for length of time according to table below
      9. Sample Master Mix Enzyme Incubation Time (hrs)
        X (A-F) X NEB 3
        Y (A-F) Y Thermo 3
        Z (A-F) Z Epi (1:10) 3
        P (A-F) X NEB 6
        Q (A-F) X NEB 12
      10. Incubate at 65C for 10 minutes;Hold at 10C until next step

      Continued tomorrow