A rapid and complete 4-step protocol for obtaining nucleotide sequence from E. coli genomic DNA from overnight cultures.
We describe a rapid and complete 4-step protocol for obtaining DNA sequence from E. coli genomic DNA starting from overnight cultures. These steps are as follows: isolation and purification of genomic DNA; PCR amplification of the desired region; purification of the amplified DNA product; and finally, direct sequencing using the dideoxy chain termination procedure with simple modifications to improve signal intensity. Clean DNA sequence can be obtained from a large number of strains in a matter of days, using easily available kit-type reagents. This protocol should be generally applicable to genomic DNA from other species.
Seetharam
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Du Pont Merck Pharmaceutical Company, Experimental Station, Wilmington, DE 19880-0328.
Dicker
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D001483:Base Sequence; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D016133:Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
D004269:DNA, Bacterial