PCR Technologies Protocols Table of Contents
Although quantitative PCR uses the same basic concept as traditional PCR, the reactions differ in that the amplicons are generally smaller and are detected indirectly using an additional dye or labeled probe or primer.
In the PCR Technologies Guide, the requisite components and quality control requirements for qPCR experiments were described in detail. With those in mind, the following is a protocol that can be used as a basic template for qPCR incorporating SYBR Green I DNA binding dye that is amenable to modification and applicable for use as validation for a set of primers. Optimization of primer concentration and annealing temperature using SYBR Green I dye are described in further detail in Primer Concentration Optimization and Primer Optimization Using Temperature Gradient, respectively.
Assays run in KiCqStart ReadyMix are optimal when using a higher primer concentration than in conventional PCR. In the protocols below, 450 nM final concentration is used. This has been observed to be the optimal concentration for severalindependent assays. However some assays may benefit from further optimization and procedures for this are described in Primer Concentration Optimization and Primer Optimization Using Temperature Gradient.
1. Place all reaction components on ice.
2. Mix and briefly centrifuge to collect contents at the bottom of the tube.
3. Prepare sufficient master mix to run all samples in duplicate.
a. Include duplicate No Template Negative Controls (NTC).
b. Calculate amount of reagents to mix. Add 10% volume to allow for pipetting error.
c. Mix well, avoiding bubbles.
4. Setup reactions:
a. For NTC reactions, add 5 μL of water to the reaction tubes.
b. For experimental reactions, add 5 μL of cDNA/gDNA solution to the reaction tubes.
c. Visually confirm that all tubes or wells contain sample at the bottom at the correct volume.
d. Carefully aliquot 15 μL of reaction master mix into each qPCR tube or plate well.
e. Cap tubes or seal the PCR plate and label (according to instrument requirements). (Make sure the labeling does not
obscure instrument excitation/detection light path.)
f. Mix reactions well and spin if needed.
5. Run samples according to the cycling protocol in Table P4‑9 and repeat the run of steps 1–3 for 40 cycles.
(Note: These conditions are specific for FAST cycling protocols).
Note: Use standard dissociation curve protocol (data collection)
6. Refer to qPCR instrument manual and Data Analysis for guidance on data analysis.