Protocol: Quantitative PCR for
Determination of Angiogenic Factors

Procedure

For best results, optimal concentrations of primers, probes, MgCl2, KCl and PCR enhancers need to be determined. Testing various combinations of primer concentrations (50-1000 nM) while keeping the probe concentration constant (250 nM) is most efficient for primer optimization. The same method could be used to optimize probe concentrations by varying probe concentrations (50-250 nM) and keeping optimal primer concentrations constant. If maximum sensitivity is not required and your PCR target is abundant, satisfactory results for probe-based qPCR are often obtained with final concentrations of both primers at 500 nM and probe at 250 nM.
1. Preparation of a reaction master mix is highly recommended to give best reproducibility. Mix all reagents but template in a common mix, using ~10% more than needed. Once template is diluted into the reaction vessel, master mix is aliquoted into the proper tube or plate for thermocycling.
Volume* Reagent Final Concentration
25µL 2X JumpStart Taq ReadyMix 1.5 units Taq DNA polymerase, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 0.2 mM dNTP, stabilizers
(0.5 µL) Reference Dye** (optional) 1x
-- µL Forward Primer 50-1000 nM
-- µL Reverse Primer 50-1000 nM
-- µL Template DNA 10 ng-100 ng
q.s. to 50 µL Water  
50 µL Total Volume  
*Volume for 50 µL reaction, however component volumes may be scaled to give the desired reaction volumes.
** Use 0.1x for ABI 7500 and Stratagene instruments; replace with FITC for BioRad iCycler.
2. Mix gently by vortexing and briefly centrifuge to collect all components at the bottom of the tube.
3. Perform thermal cycling.
Optimal cycling parameters vary with probe design and thermal cycler. Check your thermal cycler manual. It may be necessary to optimize the cycling parameters to achieve maximum product yield and/or quality.
This protocol has been used successfully with the following thermal cyclers: Stratagene MX 3000P, BioRad iCycler, and ABI 7700.
Note: Dual labeled probes are usually designed for two step 94 °C/60 °C cycling conditions. Other probes are designed for a three step regimen, with an annealing temperature of 55 °C (probe Tm ~ 60 °C). Use the cycling conditions for which your probes were designed.
  Temperature Time
Initial denaturation 94 °C 2 min.*
40 cycles:
Denaturation 94 °C 15 sec
Annealing/Extension 60 °C or 5 °C below lowest primer TM 1 min **
(Optional) Hold 4 °C - only if products will be run out on a gel
* Initial denaturation of greater than two minutes is not recommended, and is unnecessary.
** Detection is usually accomplished at this step.
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