1. Prepare PCR reaction mixture
To obtain reliable quantitative PCR reaction results, it is recommended to run three replicates for each sample. The suggested template amount is 10 ng to 100 ng for genomic DNA or 1 ng to 10 ng for cDNA template.
Please prepare the PCR reaction solution according to the list below (All reagents should be placed on ice)
| Reagent |
10 μL |
20 μL |
50 μL |
Final con. |
| SYBR Master mix (2×) |
5 μL |
10 μL |
25 μL |
1× |
| PCR Forward Primer (10 μM) |
0.2 μL |
0.4 μL |
1 μL |
0.2 μM |
| PCR Reverse Primer (10 μM) |
0.2 μL |
0.4 μL |
1 μL |
0.2 μM |
| Reference Dye (optional) |
0.2 μL |
0.4 μL |
1 μL |
1× |
| DNA |
1 μL |
2 μL |
4 μL |
|
| ddH2O |
3.4 μL |
6.8 μL |
18 μL |
|
| Total |
10 μL |
20 μL |
50 μL |
|
Notes:
a. 200 nM of primer final concentration is applicable for most cases. The concentration can be adjusted within 0.1~1.0 μM when amplification efficiency is not satisfactory.
b. Too much or too little template used may lead to inaccuracy of quantitative result. A range of 1-100 ng is recommended to result in a good Ct value (15<Ct<35). If template is stocked at high concentrations, dilute it prior to loading to prevent possible loading errors.
c. It is recommended that the amplicon length should be within the range of 100-500 bp, with 100-200 bp preferred.
d. For consistency within an experimental set, prepare a sufficient volume of reaction mix without template DNA for the DNA standard reactions and experimental sample reactions.
2. Perform quantitative PCR
Perform quantitative PCR using optimized cycling conditions. Provided below is a standard two-step program and three-step program.
Two-step PCR Program
| Step |
1 |
2 |
3 |
| Hot-Start DNA Polymerase Activation |
PCR |
Melt Curve |
|
HOLD |
40 cycles |
1 cycle |
| Denature |
Anneal/Extend |
| Temp. |
95.0°C |
95.0°C |
60.0°C |
95.0°C |
60.0°C |
95.0°C |
| Time |
5-10 mins |
15 secs |
30-60 secs |
15 secs |
60 secs |
15 secs |
| Volume |
10 μL-50 μL |
10 μL-50 μL |
Three-step PCR Program
| Step |
1 |
2 |
3 |
| Hot-Start DNA Polymerase Activation |
PCR |
Melt Curve |
|
HOLD |
40 cycles |
1 cycle |
| Denature |
Anneal |
Extend |
| Temp. |
95.0°C |
95.0°C |
50.0°C-60.0°C |
72.0°C |
95.0°C |
60.0°C |
95.0°C |
| Time |
5-10 mins |
15 secs |
30 secs |
30 secs |
15 secs |
60 secs |
15 secs |
| Volume |
10 μL-50 μL |
10 μL-50 μL |
Notes:
a. Please note that the hot-start polymerase in this system needs to be activated at 95°C for 5 minutes prior to amplification.
b. If the amplicon sequence is GC-rich, the time for pre-denaturation/enzyme activation can be prolonged to 10 minutes.
c. Extension time may be adjusted according to the qPCR instruments used. For example, the extension time should be set to no less than 30 seconds when using ABI 7700 and 7900HT, 31 seconds when using ABI 7000 and 7300, 34 seconds when using ABI 7500.
3. Attention Points in Operation (Please Read Carefully)
a. Avoid repetitive freeze-thaw cycles to prevent polymerase activity from decreasing. Aliquot the mix into small batches for frequent usage.
b. Gently invert the tube upside down several times before use. DO NOT vortex. Brief centrifugation prior to use is recommended.
c. Keep the mix from bright light during storage and usage due to the fact that the fluorescent SYBR Green I dye may fade under light over time, resulting in a decrease in performance sensitivity.
d. Due to the high sensitivity nature of the qPCR reaction, contamination of air or aerosols may lead to reaction failure or result inaccuracy. Please set up the qPCR reaction in a clean environment using filtered tips, and sterilized tubes and pipette sets.
|
Instruments do not require ROX correction
|
Roche LightCycler 480, Roche LightCyler 96，Bio-rad iCyleriQ，iQ5，CFX96, etc.
|
|
Instruments require ROX I correction
|
ABl Prism7000/7300/7700/7900，EppendorfAB Step One/Step One Plus, etc.
|
|
Instruments require ROX II correction
|
ABl Prism7500/7500Fast，QuantStudio 3 System, QuantStudio 5 System, QuantStudio6 Flex
SystemQuantStudio7 Flex System， ViiA 7 systemStratagene Mx3000/Mx3005P Corbett Roton Gene 3000, etc.
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