Mycobacterium DNA Dilution for Quantification - (Sep/18/2012 )
I will be extracting DNA from Mycobacterium tuberculosis and Mycobacterium smegmatis. At the end of the extraction I will quantify the DNA via UV Spectroscopy. To dilute the DNA for spectroscopic analysis, my protocol states "generally, a 1:50 or 1:100 dilution is adequate." (I will be using TE buffer to dilute the DNA). Can anyone recommend which dilution is best? I know if the absorbance is greater than 2.0 it means I have lipid or protein contamination in my DNA, and I would imagine not having a high enough dilution could give me a false read of above 2.0.
The read will be done at 260nm and 280nm.
Thanks a Bunch!
Basically the protocol is saying that a something in the 1:50-1:100 range works fine. You don't have to be prescriptive and use either. The dilution you should use depends on the concentration of the DNA, which you won't know until you measure it... If you are using modern equipment, such as a nanodrop, then you usually don't need to dilute your DNA at all - the software works it all put for you.
You are wrong about the absorbance by the way - ratio of 260:280 over 2 is an indicator of contamination.
Ok, thanks for the information and correcting my understanding of things.