Daniel:Notebook/HiResChrPaint/2014-1-10

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Revision as of 17:58, 13 January 2014 by >Djacobse (→‎Dye Coupling Test)
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Alu Control (Started 1/08/2014)[edit]

Back to Calendar

Production qPCR[edit]

Since I didn't make enough the first time, I'm going to do another production PCR to make more Alu probe.

1. Reaction Table
  gDNA 2X Kapa SYBR qPCR MM 100 uM AP1V6-Am 100 uM AP2V6 H20 Total Volume
per rxn 1 50 0.5 0.5 48 100
PGP1F (12.1X) 12.1 605 6.05 6.05 580.8 1210
2. Aliquot 100 uL into each of 3 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 x30
    vii. 72C 2 min
    viii. 16C hold
4. Pool 3 samples together each
5. Column Purification, elute with 40 uL EB
6. Measure concentrations in nanodrop

qPCR Results[edit]

File:2014-01-10-AluProduction.png

Nanodrop Results[edit]

Sample ALU3-A ALU3-B ALU3-C ALU3-D
ng/uL 69.6 70.3 69.9 56
ug in 40 uL 2.8 2.8 2.8 2.2
total       10.6

Much better amplification results this time. Next I'll check dye coupling results.

Dye Coupling Test[edit]

Samples

  • ALU2-AB; total 1.1 ug
  • Human Cot-1 DNA, 1 ug
0. Vaccuum centrifuge DNA; resuspend in 2.5 uL nfH2O
1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
2. Add 1.5 uL of sodium bicarbonate buffer
3. Denature samples for 5 minutes at 95C, then snap cool using ice box
4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
   *dye cannot be saved for later use.  Use immediately!
5. Add 1 uL dye to sample
6. Incubate in the dark for 1 hour
7. Add 20 uL nfH20 to sample; Measure concentration in Nanodrop
8. Centri-Sep column purification
9. Measure concentration in Nanodrop


Labeling Results[edit]

Sample ng/uL DNA pmol/uL dye pmol/uL DNA dye/probe Base:Dye Ratio (:1) DNA Capture % Dye Capture %
Alu-Pre Column 460 63 7.0 9.0 22 N/A N/A
Cot1-Pre Column 44.7 43.9 0.7 64.8 3 N/A N/A
Alu-Post Column 26.6 1.3 0.4 3.2 62 7.23 2.58
Cot1-Post Column 33.3 4.7 0.5 9.3 21 93.12 13.38