3 replies to this topic

[felixwang]

I am going to use plasmid DNA from maxi-prep to transfect cells, so I want the DNA to be as pure as possible. After ethanol precipitation, I often use ddH2O(not TE or any other buffers) to dissolve pellets. The OD260/OD280 ratio of my preps were sometimes around 1.3-1.4, but another CHCl3 and phenol extraction wouldn't help to improve the results. The interesting thing was that the OD of a certain sample is not constant. One time it could be 1.4, and the next time it would be 1.8. It is rather weired. I heard people saying that DNA in H2O ( not buffered solution) can be strange, but I don' t want to use TE or EB buffer to solve DNA because I worried that these buffers would affect the efficiency of transfection. Is there any other way to monitor the quality of DNA in ddH2O?

Another thing is that I use OD260 to decide the concentration of DNA. If it is not accurate in ddH20, how can I know the concentration?

Edited by felixwang, 30 March 2005 - 07:51 AM.

[fred_33]

hi
i think that OD260 is an accurate measure. For the ratio problem, it could be fixed by upgrading dna resuspension. In ddH2O, dna get more problems than in te buffer due to the lack of salts. Hence, you may heat dna 50° 10' to 15' and then make the measure. Heating treatment would increase greatly the resuspension process and accurate the ratio.

fred

[felixwang]

fred_33, on Mar 31 2005, 12:34 AM, said:

hi
i think that OD260 is an accurate measure. For the ratio problem, it could be fixed by upgrading dna resuspension. In ddH2O, dna get more problems than in te buffer due to the lack of salts. Hence, you may heat dna 50° 10' to 15' and then make the measure. Heating treatment would increase greatly the resuspension process and accurate the ratio.

fred

<{POST_SNAPBACK}>

Cheers Fred! Very helpful!

[Bomber]

Hey,

I wouldn´t be worried about the transfection efficiency regarding the buffers you could dissolve your dna in.
Sometimes it might be even better to use special buffer (e.g. endotoxin free buffers), well to get rid of endotoxins for example.

I could imagine that there is rather a problem with your maxiprep or anythng else than with the measure of ratio. I mean do you have to pre-warm the photometer before use for example (some older photometers still need this) and  where do store your preps? Anything cloudy in it?
I agree with fred who wrote that measure is accurate in general.

One thing to the buffers again: TE-buffer is sometimes a bit tricky due the included EDTA, which can disturb enzyme reactions after your prep. Dissolving DNA in Tris-HCl is always working fine for me.

Good luck
Cheers