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1-Step RT-PCR Master Mix

Features

  • Efficient thermostable Reverse Transcriptase and RNase Inhibitor providing high cDNA yields
  • Unique Hot Start Taq DNA Polymerase in a mix with high-quality dNTPs
  • PCR enhancers allowing sensitive low background amplification

Applications

  • One-step RT-PCR
  • Virus detection
  • Amplification of GC-rich and complex templates

Purchase 1-Step RT-PCR Master Mix

Availability: In stock Bulk requests please inquire at oem@biotechrabbit.com

Catalog # Size Package Content Price* Qty
BR0400102 100 reactions of 50 µl 2 × 1.25 ml 1-Step PCR Master Mix
2 × 125 µl RT-RI Blend
€184.00
BR0400103 500 reactions of 50 µl 5 kits BR0400102 1-Step RT-PCR Master Mix (100 rxn each)
€787.00

*Does not include applicable tax, shipping & handling, or other charges.

OR

biotechrabbit™ 1-Step RT-PCR Master Mix provides an easy and efficient way to perform both reverse transcription of RNA and PCR amplification of cDNA in one step. Only RNA template, primers and PCR-grade water are added.

The 20× RT-RI Blend, which contains a blend of an efficient thermostable reverse transcriptase and a proprietary Ribonuclease Inhibitor, ensures high yields of cDNA.

The 2X 1-Step Master Mix contains unique Hot Start Taq DNA Polymerase, dNTPs, MgCl2 and stabilizers in an optimized buffer and provides high PCR product yields with minimal background even when using low-abundance and difficult templates. PCR enhancers included in the mix allow efficient amplification of complex templates including GC or AT-rich sequences.

 

Component

Composition

1-Step Master Mix

Proprietary 2X buffer composition including Hot Start Taq DNA Polymerase, dNTPs, enhancers and stabilizers

RT-RI Blend

Proprietary 20X blend of efficient thermostable Reverse Transcriptase and unique Ribonuclease Inhibitor

 

STORAGE

-30°C to -10°C (until expiry date – see product label)

 

Quality Control
Functional Assay

One step RT-PCR using eukaryotic total RNA as a template.

 

Prevention of reaction contamination

When assembling the amplification reactions, care should be taken to eliminate the possibility of contamination with undesired DNA.

RNase contamination is an exceptional concern when working with RNA. RNase A, providing most threat to RNA integrity, is a highly stable contaminant of any laboratory. To prevent RNA from degradation and to minimize possibility of contamination One Step RT-PCR; follow the guidelines below:

  • Use separate clean areas for preparation of the samples and the reaction mixture.
  • DEPC-treat all tubes and pipette tips or use certified nuclease-free labware with aerosol filters.
  • Wear fresh gloves when handling RNA and all reagents.
  • Always assess the integrity of RNA prior to One Step RT-PCR in denaturing agarose gel electrophoresis.
  • Use only water and reagents that are free of DNA, DNAses and RNAses.
  • With every One Step RT-PCR setup, perform a contamination control reaction that does not include template DNA.
Basic Protocol
  • The mixes are designed to be used without any optimization as they have all necessary reaction components in optimal amounts for successful One Step RT-PCR.
  • Thaw on ice and mix all reagents well. Keep all reagents and reactions on ice.
  • To use time and reagents effectively, always prepare master mix for multiple reactions by mixing water, RT-RI Blend and 1-Step Mix.
  • Pipet the master mix into thin-walled 0.2 ml PCR tubes.
  • Add template and primers separately if they are not used in all reactions.

Component

Volume

Final concentration

2X 1-Step Master Mix

25 µl

20X RT-RI Blend

2.5 µl

Forward Primer (10 µM)

2 µl

0.4 µM

Reverse Primer (10 µM)

2 µl

0.4 µM

RNA Template

0.1–1 µg total RNA or

10–500 ng mRNA

 

Too much template increases the background, too low template amounts reduce the PCR accuracy

PCR Grade, RNAse-free Water

Variable

 

Total volume

50 µl

 

  • Mix and centrifuge briefly to collect the liquid in the bottom of the tube.
  • Place in the PCR cycler.
Cycling Program

Step

Temperature

Time

Cycles

cDNA synthesis

45-55°C

10–20 min

1

Initial activation

95°C

2 min

1

Denaturation

95°C

10 s

35–40

Annealing

55°C

10 s

35–40

 

Approximately 5°C below Tm of primers

Extension

72°C

30–60 s/kb

35–40

Final extension

72°C

5 min

1

 

To extend all incomplete PCR products

Storage in the cycler

4°C

Indefinitely

1

  • Add loading dye solution (see 6X DNA Loading Dye, cat. no. BR0800301) to the reactions to analyze PCR products on a gel or store them at −20°C.
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