High-yield recovery of recombinant DNA from poorly growing cosmid and lambda genomic clones.
Certain genomic sequences cannot be recovered efficiently in cosmid or lambda bacteriophage clones, presenting a barrier to 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 techniques, 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 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.
Millar
S J
SJ
Dept. of Biological Chemistry, University of Texas Health Science Center, Houston 77225.
Dempsey
D
D
Dickinson
D P
DP
D010582:Bacteriophage lambda / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D003360:Cosmids / Q000235:genetics*; D015891:Cystatins / Q000235:genetics; D004274:DNA, Recombinant / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification*; D015723:Gene Library; D005822:Genetic Vectors
D015891:Cystatins; D004274:DNA, Recombinant; D004279:DNA, Viral