Open AccessGeneration of Vector-Insensitive Dig-Labeled Probes from Large Cosmid Library Inserts Using PCR
Author(s) -
Elfed Morgan,
Julia Lodge,
J. Stephen,
Nigel L. Brown
Publication year - 1999
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/99271st02
Subject(s) - insert (composites) , cosmid , dig , microbiology and biotechnology , biology , vector (molecular biology) , southern blot , ligation , polymerase chain reaction , dna , restriction enzyme , primer dimer , genetics , gene , recombinant dna , multiplex polymerase chain reaction , mechanical engineering , engineering
We have devised a general method for producing vector-insensitive probes from clones in which insert DNA (ca. 40 kb) could not be amplified in one piece nor be excised from the vector sequence. The method involves PCR and vector-specific primers in combination with restriction digestion and ligation. It yields specific PCR products that could subsequently be labeled using DIG-11-dUTP in a single-cycle PCR. In colony blot hybridization, the probes were specific for the insert DNA from which the probe was derived and, importantly, did not detect vector sequences.