Products > PCR Products > Power Green qPCR Mix

Power Green qPCR Mix

Power Green qPCR Mix

For research use only

Components  

Component

P2101

P2102

P2103

P2104

2x Power Green qPCR mix

1ml

5x1ml

10x1ml

50x1ml

Nuclease-free Water

1ml

5x1ml

10x1ml

50x1ml

ROX Reference Dye, 100X

20μl

100μl

200μl

1000μl

 

Storage

This reagent can be stored at 4°C for 2 months and protected from light. For longer

storage, it should be kept at -20°C and protected from light.

 

Description

2xPower Green qPCR mix is designed for high-performance, high-throughput, high-specificity real-time PCR. The kit contains a Hot Start Taq DNA polymerase engineered through a process of molecular evolution. The result is a unique enzyme, specifically designed for qPCR using SYBR® Green I dye chemistry.

2xPower Green qPCR mix is a convenient premix of the components (except primers, template, and water) necessary to perform real-time polymerase chain reaction (PCR) using SYBR® Green I dye with enhanced sensitivity and specificity. The SYBR® Green dye binds to double-stranded (ds) DNA, thus providing a fluorescent signal that reflects the amount of dsDNA product generated during PCR.

 

Applications

Gene expression analysis

Low copy gene detection

Microarray validation

Gene knockdown validation


Features

• Hot Start technology with anti-Taq DNA polymerase  antibodies enables high specificity and tight reproducible amplification.

• Highly reproducible CTs over a broad dynamic range.

• Compatibility with most real-time qPCR instruments.

 

Composition of the 2x Hy-Power mix

100 mM KCl , 5 mM MgCl2, 400 μM dNTPs, 0.1 U/μl Hot Start Taq DNA Polymerase, 1x SYBR® Green and other optimized buffer components.

 

Instrument Guide Table 

Instrument

Rox Reference Dye

ABI 5700, 7000, 7300, 7700, 7900HT

Step One™,  and Step One Plus™

High Rox (2%)

ABI 7500 Rox Low  

Stratagene Mx3000P®, Mx3005P™, and Mx4000® 

Low Rox (0.4%)

Rotor-Gene™; DNA Engine Opticon™, Opticon® 2,

and Chromo 4™ Real-Time Detector;

No Rox Mastercycler® ep realplex, Smart Cycler®, Roche LightCycler® 480, Bio-Rad CFX96

No Rox

 

Protocol

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

 

1. Preparation of reaction solution

Add all the solution in a thin walled PCR tube on ice.

For a total 20μl reaction volume

Component of sample

Volume

Final concentration

2x Power Green qPCR mix

10 μl

1X

Forward Primer (10 μM)

0.4 μl

0.2 μM

Reverse Primer(10 μM)

0.4 μl

0.2 μM

Template DNA

100ng


Water, nuclease-free

to 20 μl

 

If the melting curve comes with impure peaks, customer can reduce the 2x Power Green qPCR mix amount in the reaction.

High-Sepcificity reaction system

Component of sample

Volume

Final concentration

2x Power Green qPCR mix

8 μl

1X

Forward Primer (10 μM)

0.4 μl

0.2 μM

Reverse Primer(10 μM)

0.4 μl

0.2 μM

Template DNA

100ng


Water, nuclease-free

to 20 μl

If the template is low concentration, customer can increase the 2x Power Green qPCR mix amount in the reaction.

High-Sensicity reaction system

Component of sample

Volume

Final concentration

2x Power Green qPCR mix

12 μl

1X

Forward Primer (10 μM)

0.4 μl

0.2 μM

Reverse Primer(10 μM)

0.4 μl

0.2 μM

Template DNA

100ng


Water, nuclease-free

to 20 μl

 

Note:

1). The primer concentration can be further optimized, if needed. The optimal range for primers is 0.1~1μM.

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

3) Prepare in accordance with the recommended volume for each instrument.

4)ROX Reference is required by instruments, for high ROX, add 0.2 μl ROX Reference, and for low ROX, add 0.04 μl ROX Reference.

 

2. Setup the plate

Transfer the appropriate volume of reaction mixture to each well of a PCR tube/plate. Reaction volumes may be scaled down from 20 μl to 10 μl if low volume tubes/plates are used.

Cap or seal the reaction tube/plate and centrifuge briefly.

 

3. Preform qPCR using the following thermal cycling conditions.

   2 Steps PCR Reaction (Primer Tm60)

Initial Denaturation

94

3 min*

Hold

Denature

95℃

15 sec

 

40 Cycles

Anneal/Extend

60℃

20 sec

Melting curve analysis

 

  3 Steps PCR Reaction (Primer Tm60)

Initial Denaturation

94

3 min*

Hold

Denature

95℃

15 sec

 

40 Cycles

Anneal

60℃

15 sec

Extend

72

30 sec

Melting curve analysis

*3min at 94ºC is recommended time for enzyme activation, however optimal denaturation of complex targets may require up to 3 min denaturation. 

4. Analyze the results

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

 

Important Notes

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 20μl 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 20μl qPCR reaction, use up to 2.0μl 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, to take advantage of two-step cycling. If performing real-time two-step RT-PCR, 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.

 

SYBR® Green I

2x Power Green qPCR mix contains an elevated, optimized concentration of the fluorescent dye, SYBR® Green I. High signal intensities are achieved as a result of increased tolerance to high concentrations of SYBR® Green I by the engineered, novel Hy SYBR® DNA Polymerase. SYBR® Green I binds all double-stranded DNA molecules, emitting a fluorescent signal on binding. The excitation and emission maxima of SYBR® Green I are at 494 nm and 521 nm, respectively, which are compatible with use on any real-time cycler. .

 

 

This product is only for research use only.