Biotechnology2011.01.15 18:36
Plasmid 에 insert 가 들어갔나 안 들어갔나 확인하기 위한 방법 중
흔히들 Cracking method 라고 말하는 방법이 있다.
간단히 정리하면 transformant 를 broth 배양해서 배양액을 조금 덜어 tube 에 담은 뒤,
phenol/chlroform + loading dye 가 들어있는 cracking solution 을 섞어 vortex 하여 cell 을 파쇄,
이를 원심분리하여 DNA 가 포함된 분획을 agarose gel 에 로딩하면 끝!
대략 5분 정도 걸린다.

방법은 다음과 같다.

One-step phenol/chloroform extraction method for screening recombinant plasmids by size


1. Colonies of Escherichia coli transformed with a desired vector are inoculated in 3 mL of LB medium containing appropriate amount of antibiotics.

2. After overnight growth at 37°C in a shaker, 150 μL of the saturated cultures are harvested by a 15–20-s spin at full speed in a microcentrifuge (11000×g).

3. After removing the supernatant, 40 μL of loading dye (0.1% bromophenol blue, 6% sucrose) and 14 μL of a 1:1 phenol/chloroform mixture are added to the tubes, followed by a short vortex mixing step for 5–10 s to lyse the cells.

4. The samples are subsequently centrifuged for about 3 min to separate the aqueous phase from the organic phase.

   * The bromophenol blue will be distributed equally between the two phases, and cell debris (which forms a semisolid interphase) will act as a physical barrier and prevent the disturbance of the two phases.

5. Ten microliters of the aqueous (upper) phase of each sample are loaded directly onto a 1.2% agarose gel, together with a sample of the original vector as reference.

6. After electrophoresis, the gel is stained with DNA staining dye, and nucleic acids are visualized on a UV transilluminator.

 

* Typically, about 50–100 ng of supercoiled pUC119-derived plasmid DNA, without any limitation concerning the length of the insert, can be detected using this method.

 

 

cracking 하는 조금 다른 방법들도 아래 링크를 타고 가면 확인해 볼 수 있다.

http://www.protocol-online.org/prot/Molecular_Biology/Transformation/Clone_Screening_and_others/index.html


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