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Direct Extraction of DNA from Soil for Amplification of 16S rRNA Gene Sequences by Polymerase Chain Reaction
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  • Direct Extraction of DNA from Soil for Amplification of 16S rRNA Gene Sequences by Polymerase Chain Reaction
  • Direct Extraction of DNA from Soil for Amplification of 16S rRNA Gene Sequences by Polymerase Chain Reaction
저자명
Cho. Jae-Chang,Lee. Dong-Hun,Cheol. Cho-Young,Cho. Jang-Cheon,Kim. Sang-Jong
간행물명
The journal of microbiology
권/호정보
1996년|34권 3호|pp.229-235 (7 pages)
발행정보
한국미생물학회
파일정보
정기간행물|ENG|
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이 논문은 한국과학기술정보연구원과 논문 연계를 통해 무료로 제공되는 원문입니다.
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기타언어초록

Microgram quantities of DNA per gram soil were recovered with SDS- based and freeze-and thaw procedures. The average DNA fragment size was > 23 Kb. This method generated minimal shearing of extracted DNA. However, the DNA extracts still contained considerable amounts of humic impurities sufficient to inhibit PCR. Several approaches were used to reduce the interferences with the PCR (use of CTAF in extraction step, Elutip-d column purification, addition of BSA to PCR buffer) to accomplish PCR with DNA extract as a template. Most of the DNA extracts were not digested completely by restriction endonuclease, and CTAB-TREATED ane Elutip-d column purified DNA extracts were partially digested. Regarding as restriction enzyme digestion, all PCRs failed to amplify 16S rRNA gene fragments in the DNA extracts. In the case of DNA extracts only where BSA was added to PCR buffer, PCR was successfully conducted whether the DNA extracts were treated with CTAB or purified with columns. However, these two treatments were indispensable for humic impurity-rich DNA extracts to generate the PCR-compatible DNA samples. Direct extraction of DNA, coupled with these procedures to remove and relieve interferences by humic impurities and followed by the PCR, can be rapid and simple method for molecular microbiological study on soil microorganisms.