TopoTaq DNA polymerase offers robust PCR amplification of both genomic and plasmid DNA targets directly from bacterial cultures.

Plasmid targets. DNA sequences cloned into low-copy plasmid were amplified with T7-promotor and T7-terminator primers.

16S rRNA targets. Three universal primers that specifically anneal to conservative regions encoding bacterial 16S RNAs have been employed for amplification from genomic DNA templates. The same primers were used for Escherichia coli, Klebsiella pneumoniae, various strains of Lactobacillus: bulgaricus, acidophilus, casei,  curvatus, gasseri, reuteri, sakei, delbrueckii, brevis, kefiri, plantarum, and more; also, Streptococcus thermophilus, Leuconostoc mesenteroides, Oenococcus oeni, and Pediococcus pentosaceous. Here we demonstrate PCR products obtained both from bacterial glycerol stocks and individual colonies on agar plates without DNA isolation steps. This approach can be used for typing bacteria from various media without purification of genomic DNAs. Also, the PCR products with bacteria-specific primers can be obtained directly using raw food samples.

PCR PROTOCOL Reagent Quantity per reaction Cells (glycerol stock) 1無 or 1 colony Primer mixture (10 然 each) 1 無 2X Amplification Buffer 10 無 with 6 mM MgCl2 dNTP mixture (10 mM each) 1 痞 TOPOTAQ 1U Deionized water to 20 痞 Volume 20 無

Cycling conditions: Heat at 100oC for 15 seconds Repeat the following for 30 cycles:   Denature: 100蚓 for 15 seconds   Anneal: Primer Tm - 5蚓            for 30 seconds   Extend: 68蚓 for 2 min Final step: 72蚓, 6 min Maintain the reaction at 4蚓    after cycling

PCR amplifications were carried out using the protocol above with 1無 yogurt or 1無 10% homogenized cheese or bologna