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Simple cDNA library preparation with the PrimeScript Double Strand cDNA Synthesis Kit

The PrimeScript Double Strand cDNA Synthesis Kit contains all of the reagents necessary to synthesize double-stranded cDNA from purified poly A⁺ RNA templates using the Gubler-Hoffman method (Gubler and Hoffman 1983). First-strand cDNA is synthesized using PrimeScript RT and either oligo(dT) primers or random primers. After cDNA synthesis, E. coli RNase H7 is used to nick the RNA portion of mRNA-cDNA hybrids. E. coli DNA Polymerase I is subsequently used for second-strand synthesis by nick translation, and E. coli DNA Ligase repairs breaks in the second strand. Finally, T4 DNA Polymerase is used to blunt the ends. The resulting double-stranded cDNA can be cloned into an appropriate vector to generate a cDNA library.

Cat. #	Product	Size	Price	License	Quantity	Details
6111A	PrimeScript™ Double Strand cDNA Synthesis Kit	10 Rxns	$706.00
cDNA cloning is used for structural analysis of genes and RNAs, and expression of target proteins. Generating a cDNA library involves synthesizing double-stranded cDNA that is complementary to the target mRNA, inserting the cDNA into a vector of bacterial or viral origin, and introducing the vector into bacterial or eukaryotic cells. The cloned cDNA can be then used for analysis, and in vitro transcription and translation. The PrimeScript Double Strand cDNA Synthesis Kit synthesizes double-stranded cDNA from polyA⁺RNA of animal or plant origin using the Gubler-Hoffman method. Notice to purchaser Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Documents Components You May Also Like Image Data Back Double-stranded cDNA synthesis using either oligo(dT) or random primers Double-stranded cDNA synthesis using either oligo(dT) or random primers.

Overview

Includes high efficiency PrimeScript Reverse Transcriptase to synthesize first-strand cDNA from purified poly A⁺ RNA
The resulting double-stranded cDNA can be cloned into an appropriate vector to generate a cDNA library
This kit contains positive control RNA, which can be cloned into a selectable plasmid to confirm success

More Information

Applications

Synthesis of double-stranded cDNA using the Gubler-Hoffman method (Gubler and Hoffman 1983)
Construction of cDNA libraries

Components

10 reactions

PrimeScript RT (200 U/µl)	10 µl
RNase Inhibitor (40 U/µl)	10 µl
Oligo(dT)₁₈ Primer (1 µg/µl)	20 µl
Random Primer (9 mer) (0.3 µg/µl)	20 µl
5X 1st Strand Synthesis Buffer	40 µl
dNTP Mixture (10 mM each)	40 µl
E. coli RNase H/E. coli DNA Ligase Mixture	20 µl
E. coli DNA Polymerase I (20 U/µl)	20 µl
5X 2nd Strand Synthesis Buffer	300 µl
T4 DNA Polymerase (1 U/µl)	40 µl
RNase free dH₂O	600 µl ×2
ControL RNA (1 µg/µl)*	5 µl

*The Control RNA is a 1.4-kb poly(A)⁺ RNA from the tetracycline resistance gene that can be used as a positive control. When full-length double-stranded cDNA is synthesized from this RNA template and cloned, the resulting plasmid confers tetracycline resistance.

Storage

–20°C

Reference

Gubler, U. & Hoffman, B. A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269 (1983).

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

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