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Real-Time qPCR & Reverse Transcription


Products >  Real-Time_qPCR_and_Reverse_Transcription >  MMLV_RT >  cDNA_Synthesis >  PrimeScript_ds_cDNA_Synthesis_Kit

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 polyA+ RNA templates using the Gubler-Hoffman method1. First strand cDNA is synthesized using PrimeScript RTase and either oligo(dT) primers or random primers. After cDNA synthesis, E. coli RNase H7 is used to nick the RNA portion of the mRNA/cDNA hybrid. 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.

  At-A-Glance   Documents   Images & Data

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

Components

10 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

Reference

1. Gubler, U. and Hoffman, B. J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263.


 
 
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6111A PrimeScript™ Double Strand cDNA Synthesis Kit 10 Rxns $658.00
 

 

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