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SYBR Green

Imagen ® SYBR Green qPCR Premix

Cat. NO IPQ

 

Description

Imagen ® SYBR Green qPCR Premix is a ready-to-use, 2X concentrated reagent including Hotstart Taq SYBR Green I and ROX Reference dye, specially designed for real-time PCR with intercalator method. The ROX is optimal for instruments from Applied Biosystems (models 7000,7300, 7700, 7900HT, StepOne™, and StepOnePlus™, but not Applied Biosystems 7500 Real Time PCR SystemsSmart Cycler® or LightCycler® real time instruments.) The ROX Reference dye does not participate in the PCR amplification.

 

Contents

The Imagen ® SYBR Green qPCR Premix is supplied as a ready-to-use 2x reaction mix. The formulation contains Hotstart Taq DNA polymerase, MgCl2, dNTPs, ROX Reference Dye, reaction enhancers, and stabilizers.

 

Storage

  • -20 °C
  • Protected from light
  • Avoid repeated freezing and throwing

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Reaction Mix Thawing and Handling

Imagen ® SYBR Green qPCR Premix is delivered in a 2x ready-to-use format. To use the mix, thaw the vial on ice to 4 °C.

 

Please completely mix the vial and briefly centrifuge to ensure all components are at the bottom of the tube. Store on ice protected from light until ready to use. If using automated liquid handling, let sit at ambient temperature for 10 min to further reduce the viscosity.

Application

  • Detection and Quantification of DNA targets.
  • High Throughput Applications.

 

Prepare the qPCR Reaction Mix

  1. Mix the Imagen ® SYBR Green qPCR Premix thoroughly but gently until it’s completely homogenous.
  2. Prepare the qPCR Reaction Mix for the number of reactions required as shown in table below and plus 10% overage.

Reagent

Volume (ul)

Final conc.

Imagen ® SYBR Green qPCR Premix

12.5

1x

Forward Primer(10 uM)

0.75

300 - 600 nM

Reverse Primer(10 uM)

0.75

300 - 600 nM

DNA Template

2

100 ng - 10 pg

Nuclease-free water

9

-

Final volume

25

-

 

 

 

 

 

 

 

 

 

 

  1. Vortex the tube to mix the contents thoroughly, then centrifuge briefly to collect the contents at the bottom of the tube. (*Use good pipetting practice to ensure assay precision and accuracy of dispensing.)
  1. Add DNA (and nuclease-free water, if needed) to the PCR tubes or wells containing the reaction mix, seal tubes or wells with flat caps or optically transparent film, and gently vortex to ensure thorough mixing of the reaction components.
  2. Program the thermal cycling protocol on the real-time PCR instrument.

Step

Temp. ℃

Time

Cycles

DNA polymerase activation and template denaturation

Amplification

95℃

10 min

1

Amplification

 

Template denaturation

 

95℃

20 sec

35-40

 

Annealing / Extension and plate read

58 - 62℃

60 sec   © Data acquisition

Melt Curve

95℃

60℃

95℃

20 sec

20 sec

15 sec

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  1. Load the PCR tubes or plates onto the real-time PCR instrument and start the qPCR run program.
  2. When thermal cycling is complete, perform data according to the instructions in the instrument-specific software.