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. 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.
Microbial Molecular Genetics and Cell Biology Group, School of Biological Sciences, University of Birmingham, Edgbaston, West Midlands, UK. e.morgan.20@bham.ac.uk
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E
Morgan
J M
JM
Lodge
J
J
Stephen
N L
NL
Brown
D015139:Blotting, Southern; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics*; D015342:DNA Probes / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D003856:Deoxyuracil Nucleotides; D054306:Dideoxynucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives; D015723:Gene Library; D007202:Indicators and Reagents; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012486:Salmonella typhimurium / Q000235:genetics
D015342:DNA Probes; D004269:DNA, Bacterial; D003856:Deoxyuracil Nucleotides; D054306:Dideoxynucleotides; D007202:Indicators and Reagents; C070581:digoxigenin-11-2',3'-dideoxyuridine 5'-triphosphate; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C029792:GTCGAC-specific type II deoxyribonucleases; D004076:Digoxigenin