An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends.
For the preparation of gene libraries, DNA from lambda EMBL3 phage was digested with SalI and EcoRI, and the cohesive ends partially filled-in by addition of dTTP, dCTP and Klenow fragment of DNA polymerase I (PolIk). Genomic DNA was cleaved partially with Sau3A and subsequently incubated with dATP, dGTP and PolIk. The phage and genomic DNAs were then mixed and ligated. The recombinant DNAs were packaged in vitro. The efficiency of packaging was 10(5)-10(6) of infectious phage lambda particles per microgram of the genomic DNA (as compared to approx. 10(7) per microgram for the wild-type lambda DNA). This procedure is very rapid and requires only microgram quantities of genomic DNA for preparing an entire gene library. The other important advantage is that multiple independent insertions of genomic DNA cannot occur in a single recombinant phage and self-ligation of phage DNA is blocked. It is also applicable for other SalI-containing vectors.
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D004256:DNA Polymerase I / Q000378:metabolism; D004262:DNA Restriction Enzymes; D005796:Genes; D005822:Genetic Vectors
D004247:DNA; D004256:DNA Polymerase I; D004262:DNA Restriction Enzymes