Re: [問題] 純化病毒DNA

看板Biotech (生命科學)作者aklim (我搞砸了....)時間19年前 (2007/06/29 10:34)推噓0(0推 0噓 0→)

留言0則, 0人參與討論串2/2 (看更多)

很好奇...是大學部還是研究生？技術原理的話.... 你可以研究你protocol step. 1. 5. 6. 7. 就行了.... ※ 引述《okinawaboy (歐奇娜娜)》之銘言： : 請問純化病毒DNA的原理是什麼?? : 想要咕狗一下可是都是生技公司的報價單= = : 麻煩高手們幫忙解說一下感謝 : ps:我承認是要做報告用的>"< 以下是實驗步驟 : 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 (保持無菌狀態) : 11. Resuspend viral DNA with 30μl sterilized ddH2O and count DNA conc. with OD260 : 麻煩了感謝 -- ※ 發信站: 批踢踢實業坊(ptt.cc) ◆ From: 218.175.152.83