Plasmid DNA mini-prep.(purification) - 전용 Kits 사용 방법 요약

플라스미드(Plasmid) DNA prep. Kits 란?

 플라스미드 DNA를 이용하기 위해서는 박테리아 (E. coli) 에서 분리 정제하는 과정이 필요하고, 이러한 과정을 plasmid DNA preperation (Prep.) 혹은 purification 이라고 하며, 이를 위한 prep. Kits 를 국내외 다양한 회사(promega, invitrogen, Qiagen, GeneAll, CosmoGenetech 등)에서 판매하고 있으며, 상세한 방법은 각 제품마다 조금 차이가 있지만 전반적인 방법에 대해 소개하고자 합니다.

 

플라스미드 DNA는 박테리아 및 일부 다른 유기체의 염색체 DNA와 분리된 일종의 원형 이중 가닥 DNA 분자입니다. 염색체 DNA와 독립적으로 복제할 수 있는 플라스미드 DNA는 유전 연구 및 생명 공학에 중요한 도구이며 박테리아에서 자연적으로 발생하는 것은 항생제 내성의 확산과 미생물 군집의 진화에 중요한 의미가 있습니다.

 

Reagents; Kits에 포함되어 있음 

Solution I 

Solution II

Solution III

EB (elution buffer) / * TE Buffer - 10mM Tris (pH 8.0) / 1mM EDTA (pH8.0)

PW or NW or Washing buffer

column

RNase A, indicator, etc

 

Sample: LB medium containing Antibiotics (ampicillin 50~150㎍/㎖) 1~10㎖

               ==> E.coli (including vector) incubate with shaking at 37℃ for 8-12 hr

 

Procedure

(1) Pick colony from plate by sterilized tooth pick and put into 15 or 50 ml cornical tube with 2.5ml or 5.0ml of media, respectively. et them grow till media get thick (usually overnight)

(2) Take about 1.5ml into1.5ml microtube.

(3) Centrifuge 8,000~10,000 rpm for 30 seconds.

(4) Discard medium and keep the pellet.( try to remove all the media)

 

(5) Resuspend pellet completely with 250㎕ resuspension solution(sol1) by pipeting or vortexing.

(6) Add 250㎕ of lysis solution(sol 2) and mix immediately by inverting tubes (※do not vortex) for 1-5min.

     *Make sure the mixture gets clear, which indicates that cells are totally disrupted.

(7) Add 350㎕ of neutralization solution (sol 3) and mix immediately by inverting tubes for 1-5 minutes.

(8) Centrifuge 12,000rpm for 10min.

(9) Pipet the supernatant (leave out the white pellet) and transfer into column on new microtube.

    * Make sure to shake resin sol before use to resuspend resin.(using promega)

       Add 1ml of resuspended resin sol.

       Leave them for 1 min.

(10)  Apply vaccumn or centrifuge (12,000 rpm, 1 min) to remove supernatant.

(11) Wash the column with 750㎕ of column washing solution.

(12) After all the washing solution were removed from the column, take the columns and centrifuge 4,000-8,000rpm for 20 seconds to get rid of all the washing sol.

 

(13) Take the columns and put on new microtubes, then add 50㎕ of d-H2O to column and leave them RT for 1-5 min. (or using prewarmed DW at 40~50℃)

(14) Centrifuge 8,000 ~12,000 rpm for 40~60 seconds.

(15) Remove columns and label the tubes.

     *Purified DNA in d-H20: stable at 4℃ at least for 6month -> 1yr,

    *Average yield of DNA ; 5-10㎍ total/miniprep)

 

plasmid DNA prep. kits procedure

 

 

 

DNA 정량 방법 및 계산은 링크(클릭) 참고하세요. ^^

 

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