Products > PCR Products > Power Green qPCR Mix

Power Green qPCR Mix


Power Green qPCR Mix

For research use only

Components  

Component

P2101

P2102

2× Power Green qPCR Mix

1 ml

1 ml × 5

Nuclease-free Water

1 ml

1 ml × 5

ROX Reference Dye, 100×

40 μl

200 μl

 

Storage

This reagent can be stored for 2 months at 4°C and protected from light. For longer storage, it should be kept at -20°C and protected from light.

 

Description

Power Green qPCR Mix is a ready-to-use, 2× concentrated mix that contains all the reagents (except template and primers) needed for the real-time qPCR in the SYBR Green I detection format. This product is compatible with most manufacturers' real-time fluorescent quantitative PCR instruments such as Applied Biosystems, Roche, Bio-Rad, Eppendorf, Corbett and so on.

The Hotstart Taq DNA Polymerase in the mix is chemically modified, and it can reduce non-specific amplification more effectively than the antibody-modified polymerase. At the same time, the mix contains aptamer-based inhibitors, which reduce the generation of the primer dimer and secondary metabolites. When the temperature below 45°C, the activity of the modified polymerase will be strongly suppressed, only when the temperature reached 72°C, it can be activated. This dual hot-start technique brings greater efficiency and specificity to PCR.  

 

Applications

Gene expression analysis

Low-copy gene detection

Microarray validation

Gene knockdown validation

 

Features

Exceptional specificity with dual hot-start mechanism

Tight reproducibility in Ct values over a broad dynamic range

Universal instrument compatibility

 

Table of Instrument Guide  

Instrument

ROX Reference Dye

(final concentration)

ABI® PRISM® 7000, 7700, 7900HT, ABI® 7300 qPCR Systems, GeneAmp® 5700, StepOne™, and the StepOnePlus™

High ROX (2%)

ABI® 7500 qPCR Systems, ViiA™ 7, QuantStudio™ 12K Flex, Agilent Mx3000P™ Mx3005P™ and Mx4000™

Low ROX (0.2%)

BioRad iCycler MiniOpticon, Opticon 2, Chromo 4, iQ5; Roche LightCycler 480; MJ Research DNA Engine Opticon 2, Chromo 4; Corbett Rotogene 3000, 6000

No ROX

Note: For high-ROX instrument, add 40 μl ROX Reference Dye (100×) to 1 ml 2× Power Green qPCR Mix. For low-ROX instrument, add 4 μl ROX Reference Dye (100×) to 1 ml 2× Power Green qPCR Mix.

 

Protocol

Note: Please follow the procedures outlined in the manual of each respective instrument.

 

1. Preparation of reaction solution

Add the following reagents to the proper thermal cycler reaction tube or plate on ice:

Component

Volume

Final concentration

2× Power Green qPCR Mix

10 μl

Forward Primer (10μM)

0.4 μl

0.2μM

Reverse Primer(10μM)

0.4 μl

0.2μM

Template DNA

variable

0.05-5ng/μl

Water, nuclease-free

to 20 μl

Note:

• The primer concentration can be further optimized. The optimal range for primers is 0.1~1μM.

Prepare according to the recommended volume of each instrument. 

Use 1-10ng cDNA or 10-100ng gDNA for each reaction.

 Users can increase the amount of the the qPCR Mix when using low-copy gene as template.

 Users can reduce the amount of the qPCR Mix, if the melting curve comes with impure peaks.

 

2. Setup the plate

Transfer the reaction mixture to PCR tubes/plates. Reaction volumes can be reduced to 10 μl if the instrument supports a low volume system.

Cap or seal the reaction tubes/plates then centrifuge briefly to spin down the contents and eliminate any air bubbles.

 

3. Preform qPCR using the following thermal cycling condition

Set the thermal cycling conditions using default PCR thermal cysling conditions specified in the following tables according to the instrument cycling parameters and melting temperatures of the specific primers. 

 

Standard 3-step PCR mode: 

Initial Denaturation

95ºC

20 sec-3 min*

Holding Stage

Denaturation

95ºC

10 sec

 

Cycling Stage

40 Cycles

Annealing

60ºC

10 sec

Extension

72ºC

20 sec

Melting curve analysis

Note: *20sec at 95°C is sufficient for enzyme activation, however optimal denaturation of complex targets may require up to 3 min for denaturation.

 

Fast 3-step PCR mode (Amplicons 100-150 bp):

Initial Denaturation

95ºC

3 min

Holding Stage

Denaturation

95ºC

5 sec

 

Cycling Stage

40 Cycles

Annealing

60ºC

5 sec

Extension

72ºC

5 sec

Melting curve analysis

 

Fast 3-step PCR mode (Amplicons 150-300bp):

Initial Denaturation

95ºC

3 min

Holding Stage

Denaturation

95ºC

5 sec

 

Cycling Stage

40 Cycles

Annealing

60ºC

5 sec

Extension

72ºC

10 sec

Melting curve analysis

Note:

Power Green qPCR Mix could be used for fast 3-step PCR. And the reaction time could be less than 2-step PCR that using the Mix of other brands.

Power Green qPCR Mix contains inhibitors that inhibit the Taq polymerase’s activity when under 60°C, and standard 3-step PCR has better stability and repeatability. So 3-step procedure is recommended rather than 2-step. Customers should confirm that the annealing temperature is above 60℃ when running 2-step PCR. 

  

4. Analyze the results

Data analysis varies depending on the instrument used. Please refer to your instrument user guide for information.

 

Important Notes

Hotstart Technology

Power Green qPCR Mix uses updated hot-start technology of chemical modification. It performs excellently in specificity and reproducibility. Comparing with aitibody-modified hot-start, it comes with zero animal DNA pollution.

 

Template

Genomic DNA, plasmid DNA, or cDNA can be used as template. For optimal quantitative results use up to 20ng of genomic DNA or plasmid DNA per 20 μl reaction (for smaller volumes, the amount of template should be decreased equivalently). Using greater amounts of template may reduce the maximum fluorescence signal and linearity of standard curves due to binding of the SYBR® Green I dye to the template. For two-step RT-PCR, use either undiluted or diluted cDNA generated from up to 1μg of total RNA. The volume of the cDNA added (from the RT reaction) should not exceed 10% of the final PCR volume (e.g., for a 20 μl qPCR reaction, use up to 2.0 μl of undiluted cDNA). 

 

Primers

Careful primer design and purification (HPLC-purified primers are recommended) is particularly important in order to minimize loss in sensitivity due to the production of nonspecific amplification products in SYBR® Green I-based qPCR. This effect becomes more prominent at low target concentrations. To maximize the sensitivity of the assay, use the lowest concentration of primers that can be used without compromising the efficiency of the PCR reaction (50-400nM of each primer). For optimal results, design primers that amplify PCR products 60-400 bp in length. The primers should exhibit a melting temperature (Tm) of approximately 60ºC. We recommend designing primers specifically for amplification of cDNA derived from mRNA. This prevents amplification of contaminating genomic DNA and inaccurate quantification of mRNA.

 

Melting Curve Analysis

Following real-time qPCR, melting curve analysis should always be performed to identify the presence of primer-dimers and analyze the specificity of the reaction. Program your thermocycler according to the instructions provided.

 

Quality Control

The absence of endodeoxyribonucleases, exodeoxyribonucl- eases and ribonucleases is confirmed by appropriate quality tests. Functionally tested in amplification of a single-copy gene from human genomic DNA.

 

Product Use Limitations

Power Green qPCR Mix is sold exclusively for research purposes and in vitro use. Neither the product, nor any individual components, was tested for use in diagnostic applications or for drug development, nor is it suitable for administration to humans or animals. Please refer to the MSDS, available upon request.