Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot4073

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Construction of cDNA Libraries

Stage 3: Methylation of cDNA

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 15% of the full text of this article appears below.


INTRODUCTION

The goal of this stage is to introduce methyl groups that will modify and protect naturally occurring EcoRI sites in the double-stranded cDNA. Note that Stage 3 is obligatory if synthetic linkers are used to attach the cDNA to a vector. However, the entire stage should be omitted if adaptors are used.


MATERIALS

10x EcoRI buffer

recipe 10x EcoRI methylase buffer (Optional)

recipe cautionS-Adenosylmethionine

New England Biolabs provides a solution of S-adenosylmethionine when EcoRI methylase is purchased.

EcoRI

EcoRI methylase

New England Biolabs sells an enzyme isolated from a strain of . . . [Full Text of this Article]


METHOD


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