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Biotechrabbit

[Biotechrabbit] 1-Step RT-PCR Master Mix

Cat-No. BR0400102

BR0400102 - 100 reactions of 50 µl
BR0400103 - 500 reactions of 50 µl

Biotechrabbit은 바이오의약품 및 생명과학 연구를 위한 고급 실험 재료와 서비스를 제공하는 회사입니다.

제품 설명

1-Step RT-PCR Master Mix

제품 번호

BR0400102 - 100 reactions of 50 µl

BR0400103 - 500 reactions of 50 µl

제품 특징

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

Description

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.

Other Information

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.

Protocols

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	1×
20X RT-RI Blend	2.5 µl	1×
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 T_m 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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Biotechrabbit - Official Distributor in South Korea "Morebio" 한국 공식 대리점 "모아바이오"

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