OneStep RT-PCR KIT

ORDERING INFORMATION

Catalog #	Product	Number of reactions/package	Price (EUR)
#	OneStep RT-PCR Kit	50
#	OneStep RT-PCR Kit	100

DESCRIPTION

OneStep RT-PCR Kit is a product for easy reverse transcription and PCR in “one step” with optimal recombinant of Reverse transcriptase and Hot Start DNA polymerase mixture. This kit has dual advantages of high efficiency for cDNA synthesis and high specificity of hot start polymerase to setup optimal PCR condition easy and convenience. Our specially developed buffer and enzyme make time and cost saving PCR set up by amplification of low-copy transcription and easy sample optimization. It is recommended to use in case of synthesis for less than 1 kb of PCR product.

ADVANTAGES

• easy, simple and variable application
• Hot start RT-PCR
• Synthesis of cDNA at 50°C
• increased efficiency in amplifying the target gene with high GC or secondary structure

APPLICATIONS

• RT-PCR for cDNA synthesis
• RNA virus detection

PRODUCT COMPONENTS

Contents	50 rxn		100 rxn
OneStep RT-PCR Enzyme Mix (contains the Rovalab products Reverse Transcriptase, and Hot Start Taq DNA Polymerase)	100 µl		200 µl
5x OneStep RT-PCR Buffer	500 µl		1 ml
5x Band Doctor	1 ml		1 ml
10 mM dNTP-Mix	100 µl		200 µl
RNase-free water	1 ml		1 ml

REACTION MIXTURE

Component	Volume (in µL)
RNase-free water	add up to 30 µl
5x OneStep RT-PCR Buffer	6
dNTP-Mix (10 mM of each dNTP)	1
Forward-Primer (10 µM)	1
Reverse-Primer (10 µM)	1
5x Band Doctor	optional
OneStep RT-PCR Enzyme Mix	2
Template RNA	varable
Total volume	30

CYCLING PROGRAM

Temperature (in °C)	Time	Cycles
50	30 min	1
95	15 min	1
95	20 sec	35 – 40
AT*	40 sec	35 – 40
72	1 min/k	35 – 40
72	5 min	1

AT* : Annealing Temperature; Choose the lower T_m of  both primers;

AT = T_m – 5°C ; T_m = 2°C x (A+T) + 4°C x (G+C)

TECHNICAL INFORMATION

A. Template

• Total RNA: 100 – 300 ng
• Poly(A) RNA(mRNA): 10 – 300 ng

B. 5x Band Doctor

The use of Band doctor is not necessary in general, but helps in the amplification of DNA with high GC content and complex structures. The included 5x Band doctor can be added to the reaction mixture at the final concentration of 0.5x – 1x (3 – 6 µl for 30 µl reaction).

Reaction mixture (conc. of band doctor) for 30 µl reactions

Component Concentration of Band Doctor	Mix 1    Mix 2   Mix 3
	0x	0.5x	1.0x
5x OneStep RT-PCR Buffer	6 µl	6 µl	6 µl
10 mM dNTP-Mix	1 µl	1 µl	1 µl
Forward-Primer (10 µM)	1 µl	1 µl	1 µl
Reverse-Primer (10 µM)	1 µl	1 µl	1 µl
Template RNA	variable	variable	variable
5x Band Doctor	0 µl	3 µl	6 µl
OneStep RT-PCR Enzyme Mix	2 µl	2 µl	2 µl
RNase-free water	up to 30 µl	up to 30 µl	up to 30 µl

C. Primer Design

D. Extension Time

STORAGE

Store at -20°C.