Plasmids for recombination-based screening.

Journal Article

To facilitate recombination-based screening, we constructed the ColE1-based plasmid, pi G4, that confers chloramphenicol resistance, contains a polylinker with multiple unique restriction enzyme recognition sequences, and contains the genetic marker, supF. To facilitate recombination-based screening followed by rapid DNA sequencing, we inserted the selectable marker, supF, into each of 20 high-copy-number (hcn) pUC-derived NoC plasmids that were designed for multiplex DNA sequencing. To facilitate recombination-based screening of common cDNA libraries that often contain ColE1 sequences, we constructed a supF-carrying plasmid whose replication was driven from an R6K replicon that does not share sequence homology with ColE1. Furthermore, we incorporated a useful polylinker and increased the copy number of this plasmid to create the 4.4-kb hcn plasmid, pMAD1. Thus, these plasmids allow: (1) background-free transformation of cells by a supF plasmid carrying an antibiotic-resistance marker; (2) simultaneous performance of the recombination-based assay and DNA sequencing; and (3) screening bacteriophage cDNA libraries that contain ColE1 sequences by recombination with a supF plasmid that is not homologous to ColE1 derivatives.

Full Text

Duke Authors

Cited Authors

  • Stewart, GD; Hauser, MA; Kang, H; McCann, DP; Osemlak, MM; Kurnit, DM; Hanzlik, AJ

Published Date

  • September 30, 1991

Published In

Volume / Issue

  • 106 / 1

Start / End Page

  • 97 - 101

PubMed ID

  • 1834525

International Standard Serial Number (ISSN)

  • 0378-1119

Language

  • eng

Conference Location

  • Netherlands