I want to detect minus strand of HCV and I need a control. I tried to use in vitro transcription of my control plasmid for plus and minus strand control. However, even after two times of Turbo DNAse digestion, my control sample is contaminated with DNA. Does anybody have a suggestion and experience with this problem?
DNA digestion has - like nearly everything I know - a quantitative effect only. which is generally around 4log but may be additionally dependent on the methods you were using.
You could try to go two ways:
1.Dilute your sample after DNA digestion until you cannot detect DNA and try if you can detect RNA using the same duilution.
2. Try to go down with the plasmid concentration during transfection (using carrier DNA to keep on the total amount of DNA, if required by your transfection protocol). IN many cases expression can be assured by amounts not higher than 100ng, but this may be dependent on your transfection method.
In addition, as long as you want to detect the RNA intracellularly, wash your cells extensively, because lots of the DNA may be outside of the cells in the cell culture supernatant.
Dear Ali,
try to use the following two things.
1) Try another DNase - for example, we use RQ DNase from Promega;
2) After digestion, precipitate RNA transcript with 2M LiCl (1 hour in ice): it should not precipitate DNA at all. After precipitation wish the pellet with 70% EtOH, dry 10 min at room temp. and dissolve your RNA in a buffer. Then you may repeat the digestion with DNase (it may be that it does not work properly in your transcription buffer).
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