Method for cloning restriction fragments containing the termini of BAC inserts.
We have developed a method to isolate the termini of BAC clones. The method is based on the two unique NotI sites located approximately 300 bp on either side of the EcoRI cloning site of the BAC vector pECS-BAC4. Our strategy includes the following steps: (i) generation of Southern blots with BAC clones digested with NotI and a second restriction enzyme; (ii) identification of the termini attached to the NotI/EcoRI fragment of the BAC vector via hybridization with a probe derived from sequences located between one NotI site (left or right arm) and the cloning site; (iii) ligation of the doubly digested BAC clone (NotI and the selected second restriction enzyme) with an equally doubly digested cloning plasmid vector; and (iv) confirmation of the clone as a terminus. This strategy has allowed us to begin the construction of a contig near a common bean gene that controls resistance to a group of potyviruses.
Plyler
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Department of Horticultural Sciences, University of Florida, Gainesville 32611-0690, USA.
Vallejos
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CE
D015139:Blotting, Southern; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D015342:DNA Probes; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D015723:Gene Library; D005820:Genetic Testing / Q000379:methods; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping / Q000379:methods*
D015342:DNA Probes; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases