Daniel:Notebook/ComboLock/2017-1-20

From ZhangLabWiki
Revision as of 21:59, 20 January 2017 by >Djacobse (→‎Random Hexamer RCA)
Jump to navigation Jump to search

Version 3 Oligos (Started Thursday, Jan 13)

Back to Calendar

RCA Test-Adpaters

From yesterday's results I'm going to try the short RCA again, this time using the adapter regions as primers. I'll test using Lock0301 and Latch0301, since those should only amplify if circular product exists.

  1. Rolling Circle Amplification
    1. Prepare 8.5X Master mix according to table below
    2. Add 12.5 uL master mix to each tube
    3. Add sample to new reaction tubes according to following table
    4. Reagent uL Added Master Mix (8.5X)
      Template 5 NA
      RCA Primer (Latch0301 or Lock0301); 10 uM) 2.5 21.25
      dNTP (1 mM) 0.8 6.8
      10X Phi29 Buffer 2 17
      Phi29 1 8.5
      nfH2O 8.7 73.95
      Total 20 127.5
    5. Incubate at 37C for 30 minutes
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step
  2. Qubit Measure
    1. Mix dye to Qubit buffer; 10 uL dye in 1990 uL buffer (1:200 ratio per sample)
    2. Add 190 uL dye/buffer and 10 uL standard to standard tubes
    3. Add 199 uL dye/buffer and 1 uL sample to sample tubes
    4. Measure in Qubit

Results

Sample Name 1A 1B 1AL 1BL 4AL 4BL 1AK 1BK 4AK 4BK
Sample Condition Sample-PreRCA Sample-PreRCA Sample Sample No PO4 (PCCB) No PO4 (PCCB) Sample Sample No PO4 (PCCB) No PO4 (PCCB)
RCA Primer None None Latch0301 Latch0301 Latch0301 Latch0301 Primer6 Primer6 Primer6 Primer6
Conc (ng/uL) 0.81 1.27 1.76 2.52 2.22 2.35 0.25 0.27 0.37 0.29
Fold Change from Pre-RCA NA NA 2.2 2.0 2.7 1.9 0.3 0.2 0.5 0.2

So this doesn't show anything definitive. It seems that the RCA didn't work at all with either of these. I'm going to try hexamers, which ought to amplify anything and everything (exponentially, actually)

Random Hexamer RCA

RCA Test (again) using random hexamers.

  1. Rolling Circle Amplification
    1. Prepare 4.5X 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
      Random Hexamer (100 uM) 2.5 11.25
      dNTP (1 mM) 0.8 3.6
      10X Phi29 Buffer 2 9
      Phi29 1 4.5
      nfH2O 8.7 39.15
      Total 20 67.5
    5. Incubate at 37C for 30 minutes
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step
  2. Qubit Measure
    1. Mix dye to Qubit buffer; 10 uL dye in 1990 uL buffer (1:200 ratio per sample)
    2. Add 190 uL dye/buffer and 10 uL standard to standard tubes
    3. Add 199 uL dye/buffer and 1 uL sample to sample tubes
    4. Wait 5 minutes to allow dye saturation
    5. Measure in Qubit

Results