Detection of large cDNA inserts within crude lambda gt11 lysates: a rapid and sensitive method.
A method is presented for the isolation of bacteriophage lambda DNA and the rapid identification of large cDNA inserts within crude phage lysates. The primary screening of a lambda gt11 cDNA library with a 32P-radiolabeled cDNA probe yielded 21 putative positive clones. A phage "spot-blot" analysis was employed to quickly screen these potential recombinants. This eliminated 9 of the 21 clones as the result of false positive signals. The remaining 12 recombinant phage were amplified on agarose-based media, and phage DNA was isolated using a modified plate lysate procedure. The DNA thus obtained from these crude lysates could be easily digested with EcoRI and examined by Southern blot analysis. The resulting blot was hybridized with the same cDNA probe used in the initial screening of the library. Thus, two clones harboring the longest cDNA insert were identified from a mixed phage population and were subsequently plaque purified. The procedure is rapid, sensitive, reproducible, inexpensive and allows the processing of several clones at once without sacrificing the quality or yields of the DNA preparation. Furthermore, the method obviates the need for plaque purifying all the positives obtained from the initial screening of a cDNA library.
Asundi
V
V
Weis Center for Research, Geisinger Clinic, Danville, PA 17822.
Tyler
B
B
Dreher
K
K
D010582:Bacteriophage lambda / Q000235:genetics*; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification*; D015723:Gene Library; D005821:Genetic Techniques
D004279:DNA, Viral; D004247:DNA