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PMID: 2841642 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Enhanced recovery and restriction mapping of DNA fragments cloned in a new lambda vector.

Nucleic acids research ·Vol. 16 ·No. 14B ·1988-07-25 ·Pages 6725-36

Whittaker PA, Campbell AJ, Southern EM, Murray NE

Abstract

In this paper we describe a modification to the lambda vector EMBL3 which greatly expedites the construction of restriction maps of cloned DNA sequences. In the modified vector, EMBL3cos, all the phage coding sequences are placed to the right of the cloning sites so that the left cohesive end is separated by only 200bp, rather than 20kb (as in conventional lambda vectors), from the inserted DNA fragment. We show that reliable restriction maps can be rapidly constructed from partial digests of clones made in this vector by labelling the left cohesive end with a complementary 32P-labelled oligonucleotide. In addition, we quantify the restriction of clones containing human DNA by the McrA and McrB systems of E. coli and show that the use of Mcr- plating strains can increase the yield of recombinant phage up to tenfold, to give cloning efficiencies of greater than or equal to 10(7) pfu/microgram of human DNA.

MeSH Terms

Bacteriophage lambda/genetics Chromosome Mapping Cloning, Molecular/methods DNA Restriction Enzymes/metabolism Genetic Vectors Humans Methylation Transformation, Genetic

Chemicals

DNA Restriction Enzymes

Authors & Affiliations

4 authors, click to expand affiliations / ORCID

Whittaker P A

Department of Biochemistry, University of Oxford, UK.

Campbell A J

Southern E M

Murray N E

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28 references, click to expand

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Article Info

Journal

Nucleic acids research

Abbr.

Nucleic Acids Res

ISSN

0305-1048

DOI

10.1093/nar/16.14.6725

Published

1988-07-25

Pages

6725-36

Language

English

Region

England

NLM ID

0411011

PMCID

PMC338328

Subset

External Links

PubMed source DOI: 10.1093/nar/16.14.6725 PMC Full Text View journal details

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Enhanced recovery and restriction mapping of DNA fragments cloned in a new lambda vector.

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