Hi all,
Hope someone will answer me ad not everybody is on vacations...
I was isolating my RNA using trizol from neurons but I accidentally forgot to perform the ET-OH wash after precipitating with isopropanol.
I resuspended in 10 ul and measured concentration and got low yields, when I realised my mistake I added 10 ul more and measured again the yields were much higher I don't know why, and so were the ratios 260/280.
Any suggestion? can I proceed with retrotranscription? just through everything away and start from neurons again?
Thank you all in advance
Gilda
Better start it over again.It's RNA, u know how critaical it is to start with only the best stuff. Good luck and stick to the protocol :)
Ciao ciao.
Hi Gilda,
Definitely there will be some contaminants including salts, if you don't give a ethanol wash. However, the yield can be high as small RNA fragments will also remain in the pellet. This step is avoided many times if you plan to use the RNA preparation of small RNA analysis. So if you are sure about complete removal of salt..you may give it a try for RT reaction.
bye
thank you all for your suggestions...this is the last experiment for my paper and, of course, everything that could go wrong is going wrong...including my attention...
The RNA should be in your sample. Maybe the impurities disturb your measurement. You could precipitate the RNA again with isopropanol, and then wash with 70-75% EtOH. But you might loose some of your RNA. You could try it before you start over again. It is not much work.
In what did you dissolve the RNA? If it is water, my guess is you could evaporate the water in a speedvac and perform the wash step. I only expect that the salts will not be washed away so effectively with this procedure.
If you use this unwashed RNA, the salts and phenol left overs might have a negative effect on your reverse transcription.
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