RNA denatruring agarose gel electrophoresis- help on sample preperation - (Apr/25/2008 )

Hello,

I am trying to extract bacterial RNA from soil samples, i want to run my gel with RNA sample and DNase treated sample and using "Denaturing Agarose Gel Electrophoresis of RNA" gel procedure of Ambion. I wonder while prepraring samples how should be the volumes?

I use 8μl RNA sample for DNase treatment and the final sample volume for gel is 10 μl with the buffers i add.

While preparing the sample without DNase treatment for gel, should the RNA sample I added be the same with the DNase treated one (8μl) or should be equal to final volume (10 μl) ?

a little bit confused


alanyas

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What's the concentration??

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You should be loading the same amount (concentration) of RNA in each well. So for instance, if you're loading 10µg of RNA from your DNAsed sample, you should also load 10µg of untreated RNA. The volumes don't matter that much as long as you have the correct ratio of buffer/dye to RNA, and that you don't overload or underload your wells. (Depending how big your gel and wells are). I run fairly large size RNA gels, and usually start with 15µg of each RNA sample in a volume of 4µl (dry and resuspend if needed.) Then I add 12µl of the buffer/dye (formaldehyde, Ambion), do the denaturing steps, and load. Your amounts and volumes may be different, but that's the general idea.
Hope that helped..

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yes definately helped thank you very much

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I'm glad-- You're welcome!

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