dna contamination after turbo free dna - (May/23/2011 )
I had variability in my qRT-PCR results and my supervisor recommended me to treat my RNA samples with DNas1 because in our lab we used to isolate RNA without DNase treatment so we bought Turbo DNA free from ambion but unfortunately it didnít work. I fellow the protocol but i got very diluted RNA samples and after I run qRT-PCR I got more variability even in house keeping gene. Anyone use turbo DNA free, please tell me exactly how its work and how much RNA sample you use and what is the reaction final volume.
Thanks in advance
We use Turbo DNA Free regulary. Use like 22 ul of isolated RNA, add recomended volumes and end with 30 ul or so, so only minor dilution. The RNA usually comes from Trizol, which contains traces of RNA, but not much.
Your RNA may degrade during the procedure (do you use RNase Free water?) or there may be too much DNA. To check whether your problems are due to DNA contamination, run an RT- reaction (substitute reverse transcriptase with water in RT reaction) and see how much DNA you have in your samples.
And of course after DNAse treatment you have to measure RNA concentration and add the same amount to each RT reaction, you can't use measurements done before that.
Just a few thoughts..
thanks for your response but the protocol says that if you have more than 200 ug/ml, take 10 ug and diluted to 50 ul and then add 10% of the buffer and 1 ul of DNAse and after incubation add 10% inactivation reagent. could you please tell me exactly the volumes you use.
really appreciate your help...