Daniel:Notebook/HiResChrPaint/2013-12-18: Difference between revisions
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5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB | 5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB | ||
6. Measure concentrations in nanodrop | 6. Measure concentrations in nanodrop | ||
===qPCR Results=== | |||
[[Image:2013-12-18-ALUTest.png|500px]] |
Revision as of 20:28, 18 December 2013
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