[問題] 純化病毒DNA
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
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