For in vitro transcription we normally use high concentrations of our linear DNA. After double digestion with Dnase I and two times RNA purification, the PCR on the RNA is still positive even with 10 million dilution. Is it DNA contamination or something else. Can anybody help me in this regard. Thanks
It is not possible for a standard DNA polymerase to reverse transcribe RNA, so you must have DNA contamination. I would suggest performing an acid phenol/chloroform/isoamyl alcohol (Ambion is a good source for this) extraction of the RNA sample, followed by a sodium acetate/ethanol precipitation. Any further DNA contamination can be removed by using the DNA-free kit from Ambion.
Standard RNA extraction methods are not very good at removing all traces of DNA, but the combination of acid phenol and DNase I works very well, but do the steps in that order.
Good luck!
You may also go for DNase treatment followed by Purelink in-colum (Life tech) digested DNA removal. Its a very efficient method
Thanks, I am going to purify My RNA after 1st Dnase treatment , using acidic Pnenol/ cloroform/IAA purification and performing PCR on the eluted one and if again is positive I will perform second Dnase treatment on the column.
I agree with the mark, i think ur protocol is fine, if possible do it in set
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