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

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 sep...

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Published inNucleic acids research Vol. 16; no. 14B; pp. 6725 - 6736
Main Authors WHITTAKER, P. A, CAMPBELL, A. J. B, SOUTHERN, E. M, MURRAY, N. E
Format Journal Article
LanguageEnglish
Published Oxford Oxford University Press 25.07.1988
Subjects
Bacteriophage lambda - genetics
Biological and medical sciences
Biotechnology
Chromosome Mapping
cloning vectors
Cloning, Molecular - methods
DNA
DNA Restriction Enzymes - metabolism
Escherichia coli
Fundamental and applied biological sciences. Psychology
Genetic engineering
Genetic technics
Genetic Vectors
Humans
Methods. Procedures. Technologies
Methylation
phage lambda
Transformation, Genetic
Vectors (cloning, transfer, expression). Insertion sequences and transposons
Human
Radiolabelling
Process improvement
Escherichia coli
Method
Virus
Lambda phage group
Styloviridae
DNA
Restriction map
Bacteria
Genetic engineering
Phage lambda
Molecular cloning
Escherichieae
Vector
Enterobacteriaceae
Phage
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Summary: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.
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ISSN:0305-1048
1362-4962
DOI:10.1093/nar/16.14.6725