Daniel:Notebook/HiResChrPaint/2013-12-18: Difference between revisions

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  3. Column Purification, elute with 30 uL EB
  3. Column Purification, elute with 30 uL EB
  4. Measure concentrations in nanodrop
  4. Measure concentrations in nanodrop
==qPCR Secondary Amplification==
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|style="background-color:#D9D9D9;font-weight:bold" width="65" | H20
|style="background-color:#D9D9D9;font-weight:bold" width="65" | Total Volume
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Revision as of 22:14, 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 10 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

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

qPCR Secondary Amplification

  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 (1X) 1 50 0.5 0.5 48 100
PGP1F-B (1X) 1 50 0.5 0.5 48 100