Cat No.

: NexQ-6000
Package : 1 ml

NEXpro qRT-PCR Master Mix (probe)

Description

NEXpro qRT-PCR 2X Master Mix (Probe) is an optimized ready-touse solution for real-time quantitative RT-PCR assays, Using Taqman
probe etc..
It comprises all the components necessary to perform qRT-PCR:
MMLV (RNAse H-),Hotstart Taq DNA Polymerase, ultrapure dNTPs,
MgCl2. The user simply needs to add water, template,probe and
primers. An enhanced buffer allows for RT reaction temperatures up
to 50C. This can improve detection of more difficult targets as
higher RT temperatures reduce nonspecific priming and facilitate
melting of RNA secondary structures.
The kit includes the components necessary for performing RT-PCR
amplification, and have been successfully used to DNA amplify and
detect a variety of RNA targets.

Recommended Protocol

This standard protocol applies to a single reaction where only

template, primers, and water need to be added to the qRT-PCR 2X
Master mix. For multiple reactions, scale-up volume of reaction
components proportionally. All reagents should be thawed on ice,
gently mixed and briefly centrifuged before use.
1. Thaw reagents at room temperature. Mix thoroughly and then
place on ice immediately after thawing.
2. Assemble reaction tubes on ice whenever possible to avoid
premature, nonspecific polymerase activity.
3. The following table shows recommended component volumes:
Reaction Conditions
Component

10 l

25 l

1X

10M Forward Primer

0.2~2.0 l

0.5~5.0 l

0.1~1.0 M

10M Reverse Primer

0.2~2.0 l

0.5~5.0 l

0.1~1.0 M

Variable

Variable

Variable

1 l

1 l

as needed

up to 20 l

up to 50 l

NA

qRT-PCR Master(Probe)

Kit Contents

NEXpro qRT-PCR 2X Master Mix (Probe) ...........................1 ml

Applications

Fluorescence Probe

Real-time RT-PCR
Gene expression profiling
Gene knockdown verification
Array validation

20 l reaction 50 l reaction Final Conc.

Template RNA
Water, RNase-Free

NOTE: In general, use primers greater than 0.5 M for sensitivity

and less than 0.5 M for specificity.

Quality Control

No endonuclease activity, nicking activity, exonuclease activity, or

priming activity has been detected.

Storage Conditions

Store all components at -20C in a freezer.

Note

Do not contaminate the NEXscript qRT-PCR 2X Master Mix with

primers and template DNA used in individual reactions. Thaw and
mix all components thoroughly, spin down shortly and chill on ice.

4. Ensure reactions are mixed thoroughly by pipetting or gentle

vortexing followed by a brief spin in a microcentrifuge.
5. Optional-Overlay reactions with one-half volume PCR-grade
mineral oil when not using heated lid on thermal cycler.
6. Transfer tubes on ice into a thermal cycler pre-warmed. The
following table shows recommended cycling conditions:

PCR Conditions

Use of the ROX Reference Dye

ROX reference dye is not included in this kit and may be added to
compensate for non-PCR related variations in fluorescence. Addition
of the reference dye is optional. Optimizing the ROX dye concentration within the qRT-PCR reaction is an important aspect of setup.
Too much ROX in the qRT-PCR reaction will reduce background but
also makes a low target signal difficult to distinguish from
background.

Step

Temp (C)

Time

Cycle

Reverse Transcription

45-50

10-30 min.

Initial Denaturation

95

10 min.

Denature

95

20 sec.

Anneal

50 ~ 65

40 sec.

Extend

72

60 sec./kb

Final Extension

72

5 min.

25~ 40
1

NOTE: Cycling conditions may need to be optimized, depending on

different primer and template combinations. For example, raise
the annealing temperature to prevent non-specific primer
binding, increase extension time to generate longer PCR
products.
7. After cycling, maintain the reactions at 4C or store at -20C until
ready for analysis.

For in vitro research use only

TEL : +82-31-756-5656 FAX : +82-31-696-5453 www.nexdiagnostics.com / www.geneslabs.com