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  • PCR protocol for amplification of fliC of E.coli
    S. D. Reid and T. S. Whittam

    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.
    DNA concentration is adjusted to 300 ug - 500 ug/ml as determined by agarose gel electrophoresis.

    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:

    1 ul template added to individual rxn tube and kept on ice.
    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.

    Operated by Shannon D. Manning at Michigan State University