Quantitative PCR Protocols
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.
Equipment
- Pipettes dispensing volumes from <1 to 200 μL
- Benchtop microcentrifuge
- Quantitative PCR instrument
- Laminar flow hood for PCR set up (optional)
Supplies
- DNA/cDNA template: First strand cDNA reaction diluted 1:10 to detect a medium to highly expressed targets or 1:2 to 1:5 for rare transcripts. 10 ng to 100 ng gDNA.
- KiCqStart® SYBR® Green ReadyMix™ (KCQS00/KCQS01/KCQS02/KCQS03 – depending on instrument or alternative qPCR SYBR Green Reaction Mix, refer to qPCR selection tables (Tables P4-6A, P4-6B and P4-7).
- PCR grade water: PCR grade water (W1754 or W4502) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction.
- Forward and reverse primers diluted to working concentration (10 μM working stocks):
• Custom oligos can be ordered here.
• Predesigned gene expression primers are also available for model organisms (KiCqStart® SYBR® Green Primers, KSPQ12012).
- Sterile filter pipette tips
- Sterile 1.5 mL screw-top microcentrifuge tubes (CLS430909)
- PCR tubes and plates, select one to match desired format:
• Individual thin-walled 200 μL PCR tubes (Z374873 or P3114)
• Plates
- 96-well plates (Z374903)
- 384-well plates (Z374911)
• Plate seals
- ThermalSeal RTS™ Sealing Films (Z734438)
- ThermalSeal RT2RR™ Film (Z722553)
Method
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.
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.
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