Cannot isolate total RNA using column
Posted 22 May 2013 - 01:45 PM
I am trying to isolate total RNA from Staphylococcus strain, but every time I use the spin column, the yield is extremely low, closed to nothing. Does anyone know why?
I tried no-column preparation, then I saw big peaks with A260/A280 > 1.8. However, I need to make sure there's no DNA contamination as I need to do RNA-seq afterwards. How can I digest the DNA without the involvement of column?
Thank you very much in advance for your help!
Posted 22 May 2013 - 04:47 PM
The A260/A280 thing is a bit problematic - it depends on the pH of the solution you are measuring the absorbance in, if you use water you will get a different reading to using TE or other similar reagents due to a shift in the absorbance of the A280 peak for potential contaminants. For pure RNA in correctly made TE the ratio should be 2.0. A good explanation can be found here.
- littlepumpkin123 likes this
Posted 22 May 2013 - 08:50 PM
I was using water to dissolve the final product. I think I'll try not using DNAse and use PCR to verify the results then.