To identify the genes controlled by ursolic acid, E. coli K-12 ATCC 25404 was grown in LB medium overnight, diluted 1:100 in fresh LB supplemented with 0, 10, or 30 ug of ursolic acid/ml. The same amount of ethanol was supplemented to eliminate solvent effects. The cultures were grown to an OD600 of 0.9. The cells were centrifuged in a microcentrifuge for 15 s at 20,000 x g in Mini-Bead Beater tubes (Biospec, Bartlesville, Oklahoma) that were cooled to -80°C before sampling. The cell pellets were flash frozen in a dry ice-ethanol bath and stored at -80°C until RNA isolation. 问题一：为什麽添加乙醇可以消除溶剂效应啊？溶剂效应在这边有什麽影响？ 问题二：为什麽要先冷冻到-80度再进行离心啊？ 先冷却到-80度的用意是？是要弄破 cell 吗？还是有别的用途？ 离心前要解冻吗？不解冻能分离吗？ 问题三：stored at -80°C until RNA isolation. 请问一下 这边是什麽原理啊？为什麽一直给它冰着就可以分离 RNA 咧？ The total RNA was isolated by following the protocol of the RNeasy Minikit (Qiagen), including an on-column DNase digestion with RNase-free DNase I (Qiagen). OD260 was used to quantify the RNA yield. 23S/16S rRNA values (measured by using a 2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA) and OD260 and OD280 values were used to check the purity and integrity of RNA (RNeasy Mini handbook; Qiagen). 问题四：RNase-free DNase I 是指？没有 RNase 的 or 没有 RNase 活性的？ 我想问的差不多等同於 RNase-free water 是指 "去除 RNase 的水" 还是说水被处理过所以 RNase 不会作用之类的 问题五：rRNA value 是什麽东西啊？是用来做什麽的？ 是说用这三个方法(rRNA value、OD260、OD280)检查RNA的纯度和完整性吗？ 所以 rRNA value 主要是检验哪个？都有吗？还是纯度？还是完整性？ How it works？ 我在网路上还有看到其他的 rRNA value (如28s) ，他们的差别在哪里？ 因为本身没接触过这些实验，所以实在很多不懂的，希望各先位进能指点一下 谢谢～ -- 幸福很单纯......... 所以只有单纯的人才能够拥有 --

※ 发信站: 批踢踢实业坊(ptt.cc) ◆ From: 140.112.7.59 ※ 编辑: ejohn 来自: 140.112.7.59 (05/22 17:49)