Potassium hydroxide-ethylene diamine tetraacetic acid method for the rapid preparation of small-scale PCR template DNA from actinobacteria.
Genomic DNA extraction from Gram-positive bacteria is a laborious and time-consuming process. A rapid and convenient method was established to extract genomic DNA from a single colony as a PCR template. KOH-EDTA is used as a lysis buffer to disrupt the cell envelope, releasing genomic DNA, and Tris-HCl (pH = 4) is then added to neutralize the lysate. The lysate can be used directly as a template for PCR amplification. 16S rDNA was successfully amplified from Gram-positive bacteria from the genera of Bacillus, Streptomyces, Micromonospora, Nonomuraea, Microbispora, and Staphylococcus. Amplification of the trpB gene indicated that this method could also be applied to the amplification of functional genes. Compared to colony PCR methods without KOH-EDTA, this method is extremely fast and efficient, and it is applicable to high-throughput PCR amplifications.
Zhibin
Z
Sun
Yan
Y
Huang
Yanzhuo
Y
Wang
Yuguo
Y
Zhao
Zhongli
Z
Cui
D039903:Actinobacteria / Q000737:chemistry*; D005591:Chemical Fractionation / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry; D006878:Hydroxides / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry
D004269:DNA, Bacterial; D006878:Hydroxides; D017680:Potassium Compounds; D004492:Edetic Acid; C029943:potassium hydroxide