TopoTaq DNA Polymerase Direct PCR amplification of genomic DNA from bacterial cultures and individual colonies

Direct PCR Amplification by TopoTaq Polymerase of DNA from Bacterial Cultures

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.

Using Bacterial Glycerol Stocks

Using Bacterial Colonies on Agar

PCR PROTOCOL

Reagent Quantity per reaction

Cells (glycerol stock) 1オL 
or 1 colony 
Primer mixture (10 オM each) 1 オL 
2X Amplification Buffer 10 オL
with 6 mM MgCl₂
dNTP mixture (10 mM each) 1 オl
TOPOTAQ 1U
Deionized water to 20 オl
Volume 			 20 オL

Cycling conditions:

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

Direct PCR from Glycerol Stocks of Various Bacterial Cultures

Direct PCR from Yogurt

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

Direct PCR from Cheese and Bologna