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)
Hot Start ReadyMixes (Taq, Buffer, dNTPs, Reference Dye, MgCl2) | |||
---|---|---|---|
KiCqStart® SYBR® Green qPCR ReadyMix™, Cat. No. KCQS00 | KiCqStart® SYBR® Green qPCR ReadyMix™ Low Rox , Cat. No. KCQS01 | KiCqStart® SYBR® Green qPCR ReadyMix™ with ROX, Cat. No. KCQS02 | KiCqStart® SYBR® Green qPCR ReadyMix™ for iQ, Cat. No. KCQS03 |
Compatible Instruments: | Compatible Instruments: | Compatible Instruments: | Compatible Instruments: |
Bio-Rad CFX384™ | Applied Biosystems 7500 | Applied Biosystems 5700 | Bio-Rad iCycler iQ™ |
Bio-Rad CFX96™ | Applied Biosystems 7500 | Applied Biosystems 7000 | Bio-Rad iQ™5 |
Bio-Rad MiniOpticon™ | Fast Applied Biosystems ViiA 7 | Applied Biosystems 7300 | Bio-Rad MyiQ™ |
Bio-Rad MyiQ™ | Stratagene Mx3000P® | Applied Biosystems 7700 | |
Bio-Rad/MJ Chromo4™ | Stratagene Mx3005P™ | Applied Biosystems 7900 | |
Bio-Rad/MJ Opticon 2 | Stratagene Mx4000™ | Applied Biosystems 7900 HT Fast | |
Bio-Rad/MJ Opticon® | Applied Biosystems 7900HT | ||
Cepheid SmartCycler® | Applied Biosystems StepOnePlus™ | ||
Eppendorf Mastercycler® ep realplex | Applied Biosystems StepOne™ | ||
Eppendorf Mastercycler® ep realplex2 s | |||
Illumina Eco qPCR | |||
Qiagen/Corbett Rotor-Gene® 3000 | |||
Qiagen/Corbett Rotor-Gene® 6000 | |||
Qiagen/Corbett Rotor-Gene® Q | |||
Roche LightCycler® 480 |
Hot Start ReadyMixes (Taq, Buffer, dNTPs, Reference Dye, MgCl2) | |
---|---|
SYBR Green JumpStart Taq ReadyMix for High Throughput qPCR Cat. No. S9194 | SYBR® Green JumpStart™ Taq ReadyMix™ for Quantitative PCR, Capillary Formulation Cat. No. S1816 |
Compatible Instruments: | Compatible Instruments: |
Applied Biosystems 5700 | Roche LightCycler® 480 |
Applied Biosystems 7000 | |
Applied Biosystems 7300 | |
Applied Biosystems 7700 | |
Applied Biosystems 7900 | |
Applied Biosystems 7900 HT Fast | |
Applied Biosystems 7900HT | |
Applied Biosystems StepOnePlus™ | |
Applied Biosystems StepOne™ |
Instrument | Final Reference Dye Concentration | μL of Reference Dye (per 20 μL Reaction) |
---|---|---|
Applied Biosystems 5700 | 1× | 0.2 |
Applied Biosystems 7000 | 1× | 0.2 |
Applied Biosystems 7300 | 1× | 0.2 |
Applied Biosystems 7500 | 0.1× | 0.02 |
Applied Biosystems 7500 Fast | 0.1× | 0.02 |
Applied Biosystems 7700 | 1× | 0.2 |
Applied Biosystems 7900 | 1× | 0.2 |
Applied Biosystems 7900 HT Fast | 1× | 0.2 |
Applied Biosystems 7900HT | 1× | 0.2 |
Applied Biosystems StepOnePlus™ | 1× | 0.2 |
Applied Biosystems StepOne™ | 1× | 0.2 |
Applied Biosystems ViiA 7 | 0.1× | 0.2 |
Bio-Rad CFX384™ | not used | - |
Bio-Rad CFX96™ | not used | - |
Bio-Rad MiniOpticon™ | not used | - |
Bio-Rad/MJ Chromo4™ | not used | - |
Bio-Rad/MJ Opticon 2 | not used | - |
Bio-Rad/MJ Opticon™ | not used | - |
Cepheid SmartCycler® | not used | - |
Eppendorf Mastercycler® ep realplex | not used | - |
Eppendorf Mastercycler® ep realplex2 s | not used | - |
Illumina Eco qPCR | not used | - |
Qiagen/Corbett Rotor-Gene® 3000 | not used | - |
Qiagen/Corbett Rotor-Gene® 6000 | not used | - |
Qiagen/Corbett Rotor-Gene® Q | not used | - |
Roche LightCycler® 480 | not used | - |
Stratagene Mx3000P® | 0.1X | 0.02 |
Stratagene Mx3005P™ | 0.1X | 0.02 |
Stratagene Mx4000™ | 0.1X | 0.02 |
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.
Reagent | Target Final Concentration | Volume (μL) per Single 20 μL Reaction |
---|---|---|
2× KiCqStart SYBR Green qPCR ReadyMix | 1× | 10 |
Forward primer (10 μM stock) | 0.45 μM | 0.9 |
Reverse primer (10 μM stock) | 0.45 μM | 0.9 |
PCR grade water | - | 3.2 |
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).
FAST Cycling Conditions | Temp (°C) | Time (sec) |
---|---|---|
Initial Hot Start/denaturation | 95 | 30 |
Steps 1–3 are repeated through 40 cycles | ||
Step 1 | 95 | 5 |
Step 2 | 58 | 15 |
Step 3 | 72 | 10 |
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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