July 31, 1999
Reference: Reid, S. D., R. K. Selander, and T. S. Whittam. 1999. Sequence diversity of flagellin (fliC) alleles in pathogenic Escherichia coli. Journal of Bacteriology 181:153-160.
Primers are used at a concentration of 200 ng/ul for PCR.
Template DNA is isolated according to instructions provided in the Puregene DNA isolation kit from Gentra Systems, Inc.
displayTAQ FL DNA Polymerase: 5 units/ ul (Display Systems Biotech)
Supplied PCR reaction buffer 10X concentrate: 15 mM MgCl2
dNTPs: 1.25 mM
50 ul rxns are prepared as follows:
Buffer: 5 ul
dNTPs: 8 ul
primers (200 ng/ ul): 1 ul each
ddH2O: 33 ul
TAQ: 0.5 - 1.0 ul
Cover samples with mineral oil.
PE 480 thermal cycler: 94° C 5 min. followed by 30 cycles of: 94° C 1 min., 53° C 2 min., 72° C 3 min.
Cool and hold at 4° C.
This program requires adjustment for use with other thermal cyclers (PE 9600, 9700, others) because of differences in ramping speed.