Daniel:Notebook/HiResChrPaint/2013-12-18

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Revision as of 20:28, 18 December 2013 by >Djacobse (→‎qPCR Amplification)
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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

File:2013-12-18-ALUTest.png