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Nucleic Acids Research, 1988, Vol. 16, No. 14 6725-6736
© 1988

Articles

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

Paul A. Whittaker, Annette J.B. Campbell1, Edwin M. Southern and Noreen E. Murray1

Department of Biochemistry, University of Oxford South Parks Road, Oxford, OX1 3QU 1Department of Molecular Biology, University of Edinburgh Kings Buildings, West Mains Road, Edinburgh, EH9 3JR, UK

Received May 13, 1988. Accepted June 15, 1988.

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 ≥107 pfu/µg of human DNA.


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