Haynes Lab:Notebook/SynBERC Scholars 2013/2014/03/06

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03/06/14

  • Digital PCR - luciferase & other target genes



STRATEGY

  • Try dilution-limited PCR on two single-copy targets and luciferase (unknown copy number)
  • Determine at which dilutions the single-copy targets produce less than 100% frequency of positives in a set of 8 reactions
  • Perform PCR on luciferase at this same concentration and get comparative results
  • Calculating exact copy number will require Poisson stat's, we will develop this in the future


TARGET GENES & PRIMERS

  • GAPDH (chromosome 12) - primers were selected for ChIP-qPCR on 10/17/10 at Harvard
  • MMP12 (chromosome 11) - primers were selected for ChIP-qPCR on 10/17/10 at Harvard
  • luciferase (synthetic) - primers verified on 04/18/14 at Harvard


PRIMER VERIFICATION

  • Dr. Haynes will run PCR on DNA from luciferase-carrying HEK293 cells (provided by Brendan Fries)
  • Products will be analyzed via gel electrophoresis. Just one primer pair per gene will be used for downstream steps...
  • Following information is from Putative Pc-TF target gene primers.xlsx
  1. GAPDH A3 - #359, 360
  2. GAPDH B2 - #361, 362
  3. MMP12 A3 - #325, 326
  4. MMP12 B2 - #367, 368
  5. MMP12 C2 - #327, 328
  6. luc 1 - luc f1, luc r1 - #724, 725
  7. luc 2 - luc f2, luc r2 - #726, 727
  8. luc 4 - luc f4, luc r4 - #730, 731
Reagent Volume Mix (10x) Result: gel showed no PCR amplification! Investigate purity of sample.
Genomic DNA (~80 ng/μL) 1.0 μL ---
10 μM primer mix 1.0 10.0
2x GoTaq (clear) 25.0 250.0
dH2O 23.0 230.0
  50.0  

PCR

  • 95°C, 60 sec
  • [95°C, 15 sec; 57°C, 15 sec; 72°C 15 sec] x30
  • 72°C, 60 sec
  • 4°C ∞


DILUTION-LIMITED PCR

  • Alfonso and Zazu will run PCR on the Haynes Lab machine
  • Experiment set-up
    • Test each of the three primer pairs 8 times (one strip of reactions) on three different dilutions of DNA - 72 reactions
    • Test each of the three primer pairs once on undiluted DNA and no template (water instead of DNA) - 6 reactions
    • There will be 78 reactions total, but since you are running it on the 96-slot PCR machine, this will be done in 2 hours.
  • Do single-drop fluorimetry on all 78 samples. This should go fairly quickly with the new web cam set-up.
  • Crop all droplet images down to show just the droplet surrounded by a little black space. Make a grid of these images with nicely labeled rows and columns.



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