DNase I treated RNA... - (Sep/23/2005 )
I have a question about DNase I treated RNA.
Before I just used RNA direct to do my PCR( of course I did the cDNA synthesis before my PCR).
Now, after I use DNase I to destroy my DNA contamination, I got very bad results for my PCR.
I could't get the positive results anymore....
I am thinking maybe I should increase my RNA templates (usually I add 2 ul for my cDNA synthesis) for my PCR??
Or can anyone tell me what I can do to improve my results?
Thank you very much,
I got really crappy results when amplifying cDNA every time I treated with DNAse, whether qPCR or straight PCR; I tried 3 different DNAse suppliers
I read some tutorials on Ambion's website discussing that it is almost impossible to find a DNAse that does not have some RNase contamination
I stopped using DNAse entirely
now I always run a "no RT chromosomal DNA" control with my qPCR and everything always works fine. I prep my RNA with a Stratagene kit and I designed my primers carefully, and I have not seen any evidence of DNA carry-over from the RNA prep