Additive effects of bovine serum albumin, dithiothreitol, and glycerol on PCR.
The effects of bovine serum albumin, dithiothreitol, and glycerol on PCR were studied. PCR under standard conditions failed the amplification of an enterohemorrhagic E. coli DNA fragment when the boiled bacterial cell lysate was used as the template. The addition of either one of bovine serum albumin, dithiothreitol, or glycerol in the reaction mixture allowed the specific fragment amplification; and the optimum concentrations were as follows: bovine serum albumin, 1 mg/ml; dithiothreitol, 10 mM; and glycerol, 5%. In addition, when all of the three agents were included at the above concentrations, the PCR yield was further increased. The effect of the three-agent mixture was not the template specific. Furthermore, the mixture enabled long PCR over 20 kb when Taq DNA polymerase with 3'-5' exonuclease activity was used for the amplification. Our simple PCR method allows robust PCR independent of template purity or amplification length.
Nagai
M
M
Department of Biochemistry, Iwate Medical University School of Dentistry, Japan. mnagai@iwate-med.ac.jp
Yoshida
A
A
Sato
N
N
D000818:Animals; D002417:Cattle; D002460:Cell Line; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology*; D004357:Drug Synergism; D005784:Gene Amplification / Q000187:drug effects; D005990:Glycerol / Q000494:pharmacology*; D051379:Mice; D010006:Osteoblasts; D016133:Polymerase Chain Reaction / Q000187:drug effects* / Q000379:methods; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000494:pharmacology*
D012710:Serum Albumin, Bovine; D004247:DNA; D005990:Glycerol; D004229:Dithiothreitol