Journal: Journal of Clinical Microbiology
Article Title: Elimination of Bacterial DNA from Taq DNA Polymerases by Restriction Endonuclease Digestion
Figure Lengend Snippet: Nested PCRs using primer pairs 16F plus 16R and NR plus NF amplify a product in the absence of an added template. Lanes 1 to 5 were amplified by using Amplitaq LD (Perkin-Elmer, Cheshire, United Kingdom), lanes 6 to 10 were amplified with Amplitaq (Perkin-Elmer), and lanes 11 to 15 were amplified with Taq DNA polymerase (Stratagene, Amsterdam, The Netherlands). Lanes 1, 6, and 11 were positive controls for the outer PCRs; lanes 2, 7, and 12 were reagent controls for the outer reaction; lanes 3, 8, and 13 were positive controls for the nested reaction; lanes 4, 9, and 14 contained 1 μl of the first-round reagent control amplified with the nested primers; and lanes 5, 10, and 15 were reagent controls for the nested PCR. Lane 16 contained the molecular size marker (Promega, Wis.).
Article Snippet: Amplitaq LD from Perkin-Elmer, an ultrapure preparation, is guaranteed to contain less than 10 copies of bacterial 16S rRNA gene sequences per 2.5-μl aliquot, which was sufficient to produce a false-positive result under our conditions of amplification.
Techniques: Amplification, Nested PCR, Marker