Daniel:Notebook/ComboLock/2017-1-25

From ZhangLabWiki
Revision as of 21:38, 25 January 2017 by >Djacobse (→‎Results)
Jump to navigation Jump to search

Circularization Test (Started Monday)

Back to Calendar

Protocol-New Amplification Primers

These are new primers I designed. They are slightly adjusted from the locations of AmpF/AmpR. Hopefully there is less noise.

  1. qPCR
    1. Make qPCR master mix according to following recipe
      1. 361.2 uL nfH2O
      2. 17.2 uL AmpF-CLv3
      3. 17.2 uL AmpR-CLv3
      4. 430 uL Kapa SYBR Fast
    2. Add 48 uL master mix to each well
    3. Add 2 uL sample according to table
    4. Sample Product Amt Pre/Post RCA Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 0A 1pmol Pre A1 2 25 1 1 21 50
      Sample 0B 1pmol Pre A2 2 25 1 1 21 50
      Sample 2A 10 fmol Pre A3 2 25 1 1 21 50
      Sample 2B 10 fmol Pre A4 2 25 1 1 21 50
      Sample 4A 100 amol Pre A5 2 25 1 1 21 50
      Sample 4B 100 amol Pre A6 2 25 1 1 21 50
      Sample 6A 1 amol Pre A7 2 25 1 1 21 50
      Sample 6B 1 amol Pre A8 2 25 1 1 21 50
      Sample 0AX 1pmol Post H1 2 25 1 1 21 50
      Sample 0BX 1pmol Post H2 2 25 1 1 21 50
      Sample 2AX 10 fmol Post H3 2 25 1 1 21 50
      Sample 2BX 10 fmol Post H4 2 25 1 1 21 50
      Sample 4AX 100 amol Post H5 2 25 1 1 21 50
      Sample 4BX 100 amol Post H6 2 25 1 1 21 50
      Sample 6AX 1 amol Post H7 2 25 1 1 21 50
      Sample 6BX 1 amol Post H8 2 25 1 1 21 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
  2. 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

Results

RCA with Epicentre Phi29

Still have some left, so I'm going to test its effectiveness by trying to amplify the 1 amol sample. I'll use the original Epicentre concentration and a 10X dilution, since the Epicentre Phi29 is 10X more concentrated than NEB.

  • 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 (4.5X)
      Template 5 NA
      RCA Primer (LLRC 10 uM) 2.5 11.25
      dNTP (1 mM) 0.8 3.6
      10X Buffer 2 9
      Phi29 1 0
      nfH2O 8.7 39.15
      Total 20 63
    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step