High-yield recovery of recombinant DNA from poorly growing cosmid and lambda genomic clones
Millar, S.J.; Dempsey, D.; Dickinson, D.P.
Biotechniques 13(4): 554-6 558-60 562
1992
ISSN/ISBN: 0736-6205 PMID: 1476724 Document Number: 399473
Certain genomic sequences cannot be recovered efficiently in cosmid or lambda bacteriophage clones, presenting a barrier of efforts to construct a contiguous cloned library of a genome. We have encountered such sequences during our efforts to isolate cosmid and bacteriophage lambda clones carrying members of the human type 2 cystatin gene family. Several cosmid clones constructed in the pWE 15 vector did not survive purification, and using standard techiques, we were unable to obtain significant amounts of cosmid DNA from those clones we could purify. Similarly, several lambda bacteriophage clones constructed in the lambda DASH-TM II vector could not be purified, and those lambda clones we were able to isolate gave low titers in liquid lysates. In this paper, we describe generally applicable methods for preparing high yields of recombinant DNA from such recalcitrant cosmid and lambda clones constructed in these vectors.