Hi,
I usually isolate both DNA and RNA with Qiagen AllPrep kits without problem when frozen samples in RLT buffer.
However, I have a sample frozen in RLT buffer without previous removal of cell culture media. Is there any option to rescue the sample?
Qiagen's protocol said: "Incomplete removal of the supernatant will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the Rneasy silica-gel membrane. Both effects may reduce RNA yield."
I have tried to wash the sample with PBS after thawed, then resuspend in RLT and continue with the protocol, but it didn't work.. How can I isolate DNA of this sample?
Thank you!
Not possible as the potency of RLT was compromised by to its dilution due to excessive media/buffer.
Thanks for your response!
But.. can I precipitate DNA? and maybe purify DNA after precipitating. I don't mind if I can't "rescue" RNA, but the DNA of this sample is so important. How could I do that?
Thanks again!
By excessive solvent the potency of RLT is decreased which would not be helpful in safe storage of cells.
Yes you may try to precipitate the DNA from this.
But you must try to precipitate the DNA from whatever is left by centrifuging it on high speed (considering the cells are now lysed). check for the pellet (if there is). Add trizol to the pellet and mix it vigorously followed by 30-40 minutes of incubation at ice. After incubation add chloroform to the mixture and mix it then incubate for 10 minute followed by centrifugation for 30-40 minutes at 10000g. Now you will be able to find your RNA is aqueous layer and DNA in pellet (if the nucleases didnot digest it) follwed by loading this DNA on kit for separation or manual extration.
Hope for the best.
A chance of DNA methylation beside the effect of some hormones or sugars in the medium.Regards
Epigenetic effects have a role in modifying DNA &/or RNA of grown cells. Regards
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