Daniel:Notebook/ComboLock/2017-3-25

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Revision as of 23:47, 25 March 2017 by >Djacobse (→‎Linear Product Production)
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RCA Test

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This experiment is to test the effectiveness of RCA so that I can use the most efficient method of amplification for the cell-based experiments. This builds on the T4 experiments and the circular product production. I will be using that circular product to analyze the effectiveness of RCA.

Linear Product Production (Continued from yesterday)

  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:2017-03-25-LinearProductProduction.png

Still too much circular product present. I'll try one more time to make it linear.

Linear Product Production

This time we'll start with the circular product and chop it.

  1. Bglii digest
    1. Make the following reactions (; Do not add Bglii yet
    2. Reagent Single Rxn Vol
      Circular Product (1X) 4
      Bglii Cutter Primer (100 uM) 2
      10X Buffer 3.1 2
      Bglii 1
      nfH2O 11
      Total 20
    3. Use the following thermocycler program
      1. 5 min 95C
      2. Ramp to 50C at 0.2C/s
      3. 10 min 50C
      4. 4 hr 37C; When this cycle starts add 1 uL Bglii
      5. Heat kill with 65C for 20 min

    RCA Protocol

    1. Rolling Circle Amplification
      1. Prepare master mix according to table below
      2. Reagent Single Rxn (uL) Master Mix X+ (6.2X) Master Mix X- (6.2X) Master Mix Y+ (3.2X) Master Mix Y- (3.2X) Master Mix Z+ (3.2X) Master Mix Z- (3.2X) Master Mix P+ (3.2X) Master Mix P- (3.2X) Master Mix Q+ (3.2X) Master Mix Q- (3.2X)
        Enzyme NA NEB NEB Thermo Thermo Epi (1:10) Epi (1:10) NEB NEB NEB NEB
        Circular Product (1X) 4 24.8 0 12.8 0 12.8 0 12.8 0 12.8 0
        Linear Product (1X) 4 0 24.8 0 12.8 0 12.8 0 12.8 0 12.8
        RCA Primer (10 uM) 2.5 15.5 15.5 8 8 8 8 8 8 8 8
        dNTP (1 mM) 2 12.4 12.4 6.4 6.4 6.4 6.4 6.4 6.4 6.4 6.4
        10X Buffer 2 12.4 12.4 6.4 6.4 6.4 6.4 6.4 6.4 6.4 6.4
        Phi29 1 0 0 0 0 0 0 0 0 0 0
        BSA (10 mg/mL) 0.4 2.48 2.48 0 0 0 0 1.28 1.28 1.28 1.28
        nfH2O 8.1 50.22 50.22 25.92 25.92 25.92 25.92 25.92 25.92 25.92 25.92
        Total 20 117.8 117.8 59.52 59.52 59.52 59.52 60.8 60.8 60.8 60.8
      3. Add 16.5 uL master mix to each tube
      4. Add 2.5 uL 10 uM RCA primer and to new reaction tubes according to following rules
        1. "A" samples get LLRC
        2. "B" samples get LLRC-v03 (Mono-phosphorothiorate)
        3. "B" 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 Enzyme Incubation Time (hrs)
        X (ABC) NEB 3
        Y (ABC) Thermo 3
        Z (ABC) Epi (1:10) 3
        P (ABC) NEB 6
        Q (ABC) NEB 12
      10. Incubate at 65C for 10 minutes;Hold at 10C until next step