- Digital PCR - luciferase & other target genes
- 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
- 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
- GAPDH A3 - #359, 360
- GAPDH B2 - #361, 362
- MMP12 A3 - #325, 326
- MMP12 B2 - #367, 368
- MMP12 C2 - #327, 328
- luc 1 - luc f1, luc r1 - #724, 725
- luc 2 - luc f2, luc r2 - #726, 727
- 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 ||
- 95°C, 60 sec
- [95°C, 15 sec; 57°C, 15 sec; 72°C 15 sec] x30
- 72°C, 60 sec
- 4°C ∞
- 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.