Generation of vector-insensitive dig-labeled probes from large cosmid library inserts using PCR

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...

Ausführliche Beschreibung

Bibliographische Detailangaben
Veröffentlicht in:BioTechniques. - 1993. - 27(1999), 1 vom: 01. Juli, Seite 94-6, 98, 100-2 passim
1. Verfasser: Morgan, E (VerfasserIn)
Weitere Verfasser: Lodge, J M, Stephen, J, Brown, N L
Format: Aufsatz
Sprache:English
Veröffentlicht: 1999
Zugriff auf das übergeordnete Werk:BioTechniques
Schlagworte:Journal Article Research Support, Non-U.S. Gov't DNA Probes DNA, Bacterial Deoxyuracil Nucleotides Dideoxynucleotides Indicators and Reagents digoxigenin-11-2',3'-dideoxyuridine 5'-triphosphate 137067-07-3 Deoxyribonuclease EcoRI mehr... EC 3.1.21.- Deoxyribonucleases, Type II Site-Specific EC 3.1.21.4 GTCGAC-specific type II deoxyribonucleases Digoxigenin NQ1SX9LNAU
Beschreibung
Zusammenfassung: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
Beschreibung:Date Completed 01.09.1999
Date Revised 28.09.2018
published: Print
Citation Status MEDLINE
ISSN:0736-6205