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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 50C
  • 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 l


200 l





5x OneStep RT-PCR Buffer

500 l


1 ml

5x Band Doctor

1 ml


1 ml

10 mM dNTP-Mix

100 l


200 l

RNase-free water

1 ml


1 ml

REACTION MIXTURE

Component

Volume (in L)



RNase-free water

add up to 30 l



5x OneStep RT-PCR Buffer

6



dNTP-Mix (10 mM of each dNTP)

1



Forward-Primer (10 M)

1



Reverse-Primer (10 M)

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 Tm of  both primers;

AT = Tm 5C ; Tm = 2C x (A+T) + 4C 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 l for 30 l reaction).

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

Component

Concentration of Band Doctor

Mix 1   

Mix 2

  Mix 3

0x

0.5x

1.0x

5x OneStep RT-PCR Buffer

6 l

6 l

6 l

10 mM dNTP-Mix

1 l

1 l

1 l

Forward-Primer (10 M)

1 l

1 l

1 l

Reverse-Primer (10 M)

1 l

1 l

1 l

Template RNA

variable

variable

variable

5x Band Doctor

0 l

3 l

6 l

OneStep RT-PCR Enzyme Mix

2 l

2 l

2 l

RNase-free water

 up to 30 l

up to 30 l

up to 30 l

C. Primer Design

Primers can be designed manually or using a primer design software. It is recommended that Tm of the designed primers is above 64C, and AT above 58C.

D. Extension Time

The extension time should be 1 min/kb.


STORAGE

Store at -20C.