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. 問題一：為什麼添加乙醇可以消除溶劑效應啊？溶劑效應在這邊有什麼影響？ 問題二：為什麼要先冷凍到-80度再進行離心啊？ 先冷卻到-80度的用意是？是要弄破 cell 嗎？還是有別的用途？ 離心前要解凍嗎？不解凍能分離嗎？ 問題三：stored at -80°C until RNA isolation. 請問一下 這邊是什麼原理啊？為什麼一直給它冰著就可以分離 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). 問題四：RNase-free DNase I 是指？沒有 RNase 的 or 沒有 RNase 活性的？ 我想問的差不多等同於 RNase-free water 是指 "去除 RNase 的水" 還是說水被處理過所以 RNase 不會作用之類的 問題五：rRNA value 是什麼東西啊？是用來做什麼的？ 是說用這三個方法(rRNA value、OD260、OD280)檢查RNA的純度和完整性嗎？ 所以 rRNA value 主要是檢驗哪個？都有嗎？還是純度？還是完整性？ How it works？ 我在網路上還有看到其他的 rRNA value (如28s) ，他們的差別在哪裡？ 因為本身沒接觸過這些實驗，所以實在很多不懂的，希望各先位進能指點一下 謝謝～ -- 幸福很單純......... 所以只有單純的人才能夠擁有 --

※ 發信站: 批踢踢實業坊(ptt.cc) ◆ From: 140.112.7.59 ※ 編輯: ejohn 來自: 140.112.7.59 (05/22 17:49)