Features- Includes the high efficiency enzyme PrimeScript Reverse Transcriptase to synthesize first strand cDNA from purified polyA+ RNA
- The resulting double-stranded cDNA can be cloned into an appropriate vector to generate a cDNA library
- This kit contains a positive control RNA; double-stranded cDNA synthesized from the positive control RNA template and cloned results in a plasmid that confers tetracycline resistance
Applications- Synthesis of double-stranded cDNA using the Gubler-Hoffman method1
- Construction of cDNA libraries
Components10 reactions | PrimeScript RTase (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 polyA+ 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
Reference1. Gubler, U. and Hoffman, B. J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263.
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