Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot3310

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Construction of cDNA Libraries

Stage 4: Attachment of Linkers or Adaptors

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

The first 100 words of the full text of this article appear below.


INTRODUCTION

This stage achieves four goals: polishing the ends of double-stranded DNA, ligation of synthetic linkers or adaptors, digestion of the attached linkers to create cohesive termini, and preparing the cDNA for cloning.


MATERIALS

recipe 5x Bacteriophage T4 DNA polymerase repair buffer

recipe ATP (10 mM)

Omit ATP from the ligation reaction in Step 2 if the ligation buffer contains ATP.

Bacteriophage T4 DNA ligase

Bacteriophage T4 DNA polymerase

Do not use the Klenow fragment of E. coli DNA polymerase.

Bromophenol blue (0.25% w/v in 50% glycerol)

Control DNA

Please see note to Step 10.

recipe EDTA (0.5 M, pH 8.0)

Ethanol

Optional, please see . . . [Full Text of this Article]


METHOD


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