Products > PCR Products > Power Green qPCR Mix

Power Green qPCR Mix

Power Green qPCR Mix

For research use only

Components 

Component

P2101

P2102

2x Power   Green qPCR Mix

1 ml

5x1 ml

Nuclease-free   Water

1 ml

5x1 ml

ROX   Reference Dye, 100×

20 μl

100 μl

 

Storage

Keep the Power Green qPCR Mix away from light. If stored at -20, the product is stable for 2 years. Avoid repeated freezing and thawing. If stored at 4, the product is stable for 6 months. It can be stable for 7 days at room temperature as long as the product pakcaging is not open.

 

Description

Power Green qPCR Mix is a ready-to-use, 2x 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 , the activity of the modified polymerase will be strongly suppressed, only when the temperature reached 72 , it can be activated. This dual hot-start technique brings greater efficiency and specificity to PCR. 

 

Applications

n   Gene expression analysis

n   Low-copy gene detection

n   Microarray validation

n   Gene knockdown validation

 

Features

        Exceptional specificity with dual hot-start mechanism.

        Tight reproducibility in Ct values over a broad dynamic range.

        Universal instrument compatibility.

 

Instrument Guide Table

Instrument

ROX Reference Dye

(final concentration)

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

High ROX (1%)

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 20 μl ROX Reference Dye (100x) to 1ml 2x Power Green qPCR Mix. For low ROX instrument, add 4μl ROX Reference Dye (100x) to 1ml 2x 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

Amount

Final concentration

2x Power Green qPCR Mix

10 μl

1×

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

  

High-sensitivity reaction system

Component

Amount

Final concentration

2x Power Green qPCR Mix

12 μl

1×

Forward Primer (10 μM)

0.4 μl

0.2 μM

Reverse Primer (10 μM)

0.4 μl

0.2 μM

Template DNA

100 ng


Water, nuclease-free

to 20 μl

Note:

1) Use 1-10 ng cDNA or 10-100 ng gDNA for each reaction. Customer can increase the amount of the the 2× Power Green qPCR Mix when using low-copy gene as template.

2) Prepare according to the recommended volume of each instrument.

Customer can reduce the amount of the 2x Power Green qPCR Mix, if the melting curve comes with impure peaks.

 

2. Set up and run the real-time PCR instrument

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.

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

94℃

3 min

Holding Stage

Denaturation

95℃

10 sec

 

Cycling Stage

40 Cycles

Annealing

Tm-5℃

10 sec

Extension

72

20 sec

Melting curve analysis


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

Initial Denaturation

94℃

3 min

Holding Stage

Denaturation

95℃

5 sec

 

Cycling Stage

40 Cycles

Annealing

55℃

5 sec

Extension

72℃

5 sec

Melting curve analysis

 

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

Initial Denaturation

94℃

3 min

Holding Stage

Denaturation

95℃

5 sec

 

Cycling Stage

40 Cycles

Annealing

55℃

5 sec

Extension

72℃

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, 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.

 

3. Analyze the results

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

 

Important Notes

Hot Start 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. Excessive concentration of templates can result in the non-linearity of the standard curve, because many of the fluorescent dyes are combined with the template.

 

Primers

Best primers’ final concentration is 0.2-0.6 μM. The higher the concentration of the primer, the more efficient the expansion, but the worse the specificity. 

 

3-step PCR

Numerous tests have proven that classical 3-step PCR gets better stability and repeatability. The optimum temperature of Taq DNA Polymerase is 74℃, most qPCR extending time at 72℃ could be reduced to 5 sec. This could be even faster then popular 2-step PCR. Initial denaturation for 3 min could active Taq DNA Polymerase and make the template depolymerization completely. This will increase the templates’ sensitivity.