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