I am trying to extract RNA from bacterial cells (E.coli) grown in M9 till they reach OD 0.5.
Problems i have encountered:
1. Culture takes 6-7 hours to reach OD (M9 content "1 L" =100 ml 10xM9 salts+ 900 ml ddH20+ 1 ml sugar + 100ul CaCl2+ 2 ml MgS04+ 500ul Trace elements)
Does the lack of thiamine affect the time it take to reach OD?
2. By the time it reaches the OD, i start the RNA extraction using RNeasy mini Kit (Qiagen) , however by the time i am done, the central lab; which contains the fluorometer; is closed. So, i wait till next to know the RNA concentration to add the DNase. The RNA is kept at -20 as we don't have access to -80. When i read the concentration using fluorometer, i get out of range "too low" results.
Is fluorometer an accurate way to measure RNA? (no access to nanodrop at our facility, however we do have a spectrophotometer)
Is there a way to preserve the RNA till next day without it degrading ? Is there someway i can enhance my work protocol using the restrictions am facing?
Thank you for your help in advance.
Hi
Maybe low concentration of rna because of freeze and thow.
It seems better you freez cells and start extracting next day.
On the other hand you can use rna later buffer for freezing cells.
Good luck
Alireza, do you mean freeze the sample when reach proper OD? Could trizol be used for preservation of pellet instead of rna later buffer?
Thank you for your help
Yes, freez them at proper OD.
I have not worked with trizol so I dont have any idea.
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