An improved protocol and a new grinding device for  extraction of genomic DNA from microorganisms by a two-step extraction procedure.
Current protocols to extract genomic DNA from microorganisms  are still laborious, tedious and costly, especially for the species with thick  cell walls. In order to improve the effectiveness of extracting DNA from  microbial samples, a novel protocol, defined as two-step extraction method,  along with an improved tissue-grinding device, was developed. The protocol  included two steps, disruption of microbial cells or spores by grinding the  sample together with silica sand in a new device and extraction of DNA with an  effective buffer containing cell lysis chemicals. The device was prepared by  using a commercial electric mini-grinder, adapted with a grinding stone, and a  sample cup processed by lathing from a polytetrafluoroethylene rod. We tested  the method with vegetative cells of four microbial species and two microbial  spores that have thick cell walls and are therefore hard to process; these  included Escherichia coli JM109, Bacillus subtilis WB600, Sacchromyces  cerevisiae INVSc1, Trichoderma viride AS3.3711, and the spores of S.  cerevisiae and T. viride, respectively, representing Gram-positive  bacteria, Gram-negative bacteria, yeast, filamentous fungi. We found that this  new method and device extracted usable quantities of genomic DNA from the  samples. The DNA fragments that were extracted exceeded 23 kb. The target  sequences up to about 5 kb were successfully and exclusively amplified by PCR  using extracted DNA as the template. In addition, the DNA extraction was  finalized within 1.5 h. Thus, we conclude that this two-step extraction method  is an effective and improved protocol for extraction of genomic DNA from  microbial samples.
State Key Laboratory of Non-Food Biomass Enzyme Technology, National Engineering Research Center for Non-Food Biorefinery, Guangxi Key Laboratory of Biorefinery, Guangxi Academy of Sciences, Nanning, Guangxi Province, China.
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Zhang
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Chen
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Lu
D004269:DNA, Bacterial / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000302:isolation & purification*
D004269:DNA, Bacterial; D004271:DNA, Fungal