Daniel:Notebook/HiResChrPaint/2013-12-18
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ALU Control
I have the ALU primers in and I'm testing whether or not I can amplify working probes from genomic DNA.
Genomic DNA Extraction
I did this using PGP1F fibroblasts from my cell culture over in CERC.
1. Trypsinize cells using 1 mL Trypsin LE 2. Resuspend in 10 mL DMEM 3. Spin at 1000 rpm for 3 minutes, remove supernatant 4. Resuspend in 1 mL Lysis buffer from Zymo kit 5. Follow Zymo kit instructions for monolayer cells
Nanodrop Results
I separated the DNA into two columns, so there are two samples (A and B)
Sample | PGP1F-A | PGP1F-B |
ng/uL | 37.4 | 27.4 |
ug in 100 uL | 3.7 | 2.7 |
total | 6.5 |
Since I seem to have genomic DNA, I can now test the primers by doing a PCR run.
qPCR Amplification
This is only a test run, so I'm going to do it in triplicate (if this succeeds I'll have enough DNA to run the DpnII digestion).
1. Reaction table
gDNA | 2X Kapa SYBR qPCR MM | 100 uM AP1V61U-Amino | 100 uM AP2V6 | H20 | Total Volume | |
per rxn | 1 | 50 | 0.5 | 0.5 | 48 | 100 |
PGP1F-A (3.1X) | 3.1 | 155 | 1.55 | 1.55 | 148.8 | 310 |
PGP1F-B (3.1X) | 3.1 | 155 | 1.55 | 1.55 | 148.8 | 310 |
2. Aliquot 100 uL into each of 12 qPCR tubes 3. PCR protocol: i. PCR 3 min 95C ii. 95C 3 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii x17 vii. 72C 2 min viii. 16C hold 4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) 5. Column Purification, elute with 40 uL EB 6. Measure concentrations in nanodrop
qPCR Results
Nanodrop Results
Sample | PGP1F-A | PGP1F-B |
ng/uL | 12.9 | 13.4 |
ug in 40 uL | 0.5 | 0.5 |
total | 1.0 |
So first round PCR is not at all lucrative. This product will still be used for several tests, however. The first test will be for DpnII cleavage. I'll also check to see if second round PCR works.
DpnII Digestion
1. Reaction Table
Sample | PGP1F-A | PGP1F-B |
ng/uL | 12.9 | 13.4 |
ug in 30 uL | 0.4 | 0.4 |
uL 10X buffer | 5 | 5 |
uL DpnII (50U/uL) | 1 | 1 |
uL nfH2O | 14 | 14 |
Total | 50 | 50 |
2. Incubate samples at 37C for 2 hours; 20 minute heat shock at 65C 3. Column Purification, elute with 30 uL EB 4. Measure concentrations in nanodrop