作者aklim (我搞砸了....)
看板Biotech
标题Re: [问题] 纯化病毒DNA
时间Fri Jun 29 10:34:22 2007
很好奇...是大学部还是研究生？
技术原理的话....
你可以研究你protocol
step. 1. 5. 6. 7.
就行了....
※ 引述《okinawaboy (欧奇娜娜)》之铭言：
: 请问纯化病毒DNA的原理是什麽??
: 想要咕狗一下可是都是生技公司的报价单= =
: 麻烦高手们帮忙解说一下 感谢
: ps:我承认是要做报告用的>"< 以下是实验步骤
: Purification of viral DNA from infected cells
: 1. Collect infected cell lysate and transfer to 2ml tube→spin down at
: 1000xg for 5min at 4°C→add 1.5ml lysis buffer at 4°C for 10 min,
: rocking periodically
: 2. spin down at 1000xg for 5min at 4°C
: 3. PBS wash ( 2X, repeat (3) and (4) step ): Discard the supernatant and
: resuspend the pellet gently in 1.5ml cold PBS
: 4. Centrifuge at 1000xg for 5min at 4°C
: 5. Resuspend the pellet gently in 400μl extraction buffer + proteinase K( 1mg/ml ) and incubate at 37°C with gently mixing for overnight
: 6. Extract viral DNA with phenol-chloroform-IAA (2X)→chloroform (2X); NOTE: get supernant; cut-off tip
: 7. DNA-precipitate: add equal volume isopropanol and 1/10 volume 3M sodium acetate for 30 min at -50°C
: 8. Centrifuge for 30min at 4°C
: 9. Wash DNA pellet with 70% ethanol (2X)
: 10. Air dry at laminar flow (保持无菌状态)
: 11. Resuspend viral DNA with 30μl sterilized ddH2O and count DNA conc. with OD260
: 麻烦了 感谢
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