Contaminating insert degradation by preincubation colony PCR: a method for avoiding false positives in transformant screening.
Colony PCR is a convenient alternative to conventional plasmid isolation and restriction digestion for high-throughput screening of recombinant colonies. However, an insert carryover from the ligation mix, adhered to colony or agar plate, generates a substantial number of false positives. To avoid this, a simple single-tube technique involving pre-PCR nuclease incubation has been developed by optimizing a buffer system that provides nuclease action and PCR amplification sequentially. Results presented in this work provide a technique that is amenable for high-throughput screening of recombinant colonies.
Agrawal
Vineet
V
Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Punjab 160062, India.
Roy
Nilanjan
N
D000135:Acid Phosphatase / Q000235:genetics; D001234:Aspergillus niger / Q000201:enzymology; D004262:DNA Restriction Enzymes / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D005189:False Positive Reactions; D016133:Polymerase Chain Reaction / Q000379:methods*; D014170:Transformation, Genetic
D003851:Deoxyribonucleases; D004262:DNA Restriction Enzymes; D000135:Acid Phosphatase