An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends

Tipo de material: Texto

TextoSeries ; Gene, 42(1), p.119-123, 1986Trabajos contenidos:

Zabarovsky, E.R
Allikmets, R.L

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Resumen: For the preparation of gene libraries, DNA from iEMBL3 phage was digested with Sal1 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 105-lo6 of infectious phage 1, particles per pg of the genomic DNA (as compared to approx. 10' per pg for the wild-type 2 DNA). This procedure is very rapid and requires only pg 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 z/I-containing vectors.

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For the preparation of gene libraries, DNA from iEMBL3 phage was digested with Sal1 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 105-lo6 of infectious phage 1, particles per pg of the genomic DNA (as compared to approx. 10' per pg for the wild-type 2 DNA). This procedure is very rapid and requires only pg 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 z/I-containing vectors.

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