I am extracting the RNA from E. coli, However, the result in Gel electrophoresis shows quite like degradation even the OD 260/280 and 260/230 are very good (higher than 2. In the gel picture, I expect only two clear bands 23 S and 16 S using RNeasy Mini Kit. But here in the gel picture, I can see the 5 S RNA as well. And also has some smears.
For TRizol always has DNA contamination after doing PCR even if I digest it with DNase.
Thanks in advance for your help and suggestions.
Best Wishes
Tahera
you can do a proper clean up after Trizol extraction. kit usually yields highly pure RNA but yield is less as compared to Trizol method. you can increase DNase treatment time and check on gel any residual DNA.
Hi, I extracted RNA from brain tissue using TRizol method and mostly got two clear bands. Most important tip is always perform homogenization step on ice, temperature must be low enough. I have personal experience of a degraded RNA incase of homogenization in absence of ice. Also, I hope you are using RNase free water, if not then try with it. Rest, follow the instructions mentioned, I hope you will get a better result.
Good luck :)
Will try my best for further optimization. Yes, another problem is that the RNA is very easy to degrade.
Thank you so much for your suggestions Sadab Khan and Aamra Mahboob
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