Daniel:Notebook/HiResChrPaint/2014-1-10

From ZhangLabWiki
Revision as of 20:01, 10 January 2014 by >Djacobse (→‎Dye Coupling Test)
Jump to navigation Jump to search

Alu Control (Started 1/08/2014)

Back to Calendar

Production qPCR

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

File:2014-01-10-AluProduction.png

Nanodrop Results

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

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