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EasyScript cDNA Synthesis Kit

Item #: ASCDNART4 
EasyScript Reverse

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EasyScript cDNA Synthesis Kit

Order#

Description

Quantity

Rxn

G233

EasyScript cDNA Synthesis Kit

5000U (25uL)

25 rxns

G234

EasyScript cDNA Synthesis Kit

20,000U (100uL)

100 rxns

Application:

  • First strand cDNA synthesis for PCR
  • Construction of cDNA libraries
  • Generation of probes for hybridization

EasyScript cDNA Synthesis Kit is a complete system for the efficient synthesis of first strand cDNA from RNA templates. The recombinant RNasin Ribonuclease Inhibitor, supplied with the kit effectively protects RNA template from degradation.

The kit is also supplied with both oligo(dT) and random primers. The oligo(dT) anneals selectively on the poly(A) tail of mRNA. Random primers do not require the presence of poly(A). Therefore, they can be used for transcription of the 5'-end regions of mRNA. Gene-specific primers may also be used with the kit. The first strand of cDNA can be directly used as a template in PCR.

EasyScript Reverse Transcriptase (RTase) within the kit is a genetically modified form of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV). Both the EasyScript and EasyScript Plus are RNase H deficient (negative). The enzyme is purified from bacteria host as a single holoenzyme of 71 kDa with the capacity of first-strand cDNA synthesis of template up to 9kb and can be used for routine cDNA synthesis.

Kit Components:

Components

EasyScript cDNA Synthesis Kit

 

G233

G234

EasyScriptRTase (200U/µl)

5,000U

20,000U

Oligo(dT) (10µM)

40 µl

160 µl

Random Primers (10µM)

40 µl

160 µl

5x RT buffer

150 µl

600 µl

RNasin (40U/µl)

15 µl

60 µl

dNTP (10mM)

40 µl

160 µl

RNase-free H2O

1 ml

2x1 ml

Size

25 rxns

100 rxns

Storage Buffer:
50mM Tris-HCl (pH 8.3), 100 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 0.1% (v/v) Triton X-100, and 50% (v/v) glycerol.

Storage: 
Store at -20°C in a frost-free freezer. Multiple freezing and thawing of RNA should be avoided. Keep RNA on ice all the time. It is recommended that the first strand cDNA synthesis is carried out under conditions where RNase contamination has been eliminated.

General Protocol:

RT-PCR reactions should be assembled in a RNA-free environment. The use of "clean", automatic pipettes designated for PCR and aerosol resistant barrier tips are recommended. 
1. Thaw template RNA and all reagents on ice. Mix each solution by vortexing, and centrifuge briefly to collect residual liquid from the sides of the tubes.
2. Prepare the following reaction mixture in a PCR tube on ice:

 

Volume

Concentration (final 20µl)

Total RNA, or poly(A)+RNA

Variable

0.5-5µg per reaction

 

 

50ng-0.5µg per reaction

Oligo(dT) (10µM)

1µl

0.5µM

or Random Primer (10µM)

1µl

0.5µM

or Sequence-specific Primer

Variable

10-15pM

dNTP (10mM)

1µl

500µM

5X RT Buffer

4µl

1X

RNasin (40U/µl)

0.5µl

20U per reaction

EasyScript RTase (200U/µl)

1µl

200U per reaction

RNase-free H2O

Variable

-

Final volume

20µl

-

3. Incubate at 25°C for 10 minutes if random primer is used. Omit this step if Oligo(dT) primer or sequence specific primer are used.
4. Incubate the mixture at 42°C for 60 minutes.
5. Stop the reaction by heating at 85°C for 5 minutes.
6. Chill on ice. The newly synthesized first-strand cDNA now can be used directly for PCR amplification.

Notes:
1. Isolation of poly(A)+ RNA from total RNA is not mandatory; however, doing so may improve the yield and purity of the final product.
2. RNA sample must be free of contaminating genomic DNA.
3. Unlike the oligo(dT) priming, which usually requires no optimization, the ratio of a random primer to RNA is critical in terms of the average length of cDNA synthesized in the reaction. Increasing the ratio of random primer/RNA will result in higher yield of shorter (~500 bp) cDNA, whereas decreasing this ratio will produce longer products.

4. The synthesized cDNA should be stored at -20°C.

PNCDNART

Pkg
Kit
 
SKU Number Pkg Vol Price Quantity Add to Cart
G234 Kit 100 rxns
415.80
Each
EasyScript Reverse
EasyScript cDNA Synthesis Kit
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