Hi

What is the best way to measure plasmid DNA concentration using spectrophotometer.
should I do different dilutions to make sure is in the right absorbance range?
Should I dilute it in H2O or TE buffer?

which ratio is more relevant in terms of DNA quality 260/280 or 260/230?

Hi!

1) Try and see whats happening
2) Depends on your blank-sample; DNA is considered to be more stable in TE, though EDTA might disturb subsequent in enzymatic reactions in few cases
3) 260/280 probably more relevant, anyway 260/230 shouldn´t be out of range; is important as well.

Good luck
Cheers

hi
generally i use two tubes of same dilution for confirmation. and i think accurate measures are when DO is around 0.2 at 260.
I dissolve plasmids in H20. But since i saw my miliQ was at ph 5 i disolve it in 10mM tris pH 8 and it works well. Also i agree with bomber and think EDTA may interfers with though reactions as ligation.
260/280 ratio is better to see purity of your fragment.
finally best way to measure is to do a spectrum ranging 230 290nm.

Hi, macedo.
I usually dilute my sample 40X in sterile miliQ water, and it works fine. If there is enough sample volume, then duplicate of the dilution will be done. When taking the absorbance, I will take 2-3 readings and get the average. A260/A280 is quite accurate in determining the purity of the DNA.
Good luck.

HI, I generally dilute 1ul DNA in 1ml MillQ, and take readings as first Initial another after waiting for 2minutes. Sometimes u find variation if you wait. If it approximately correlates with what u see on gel I continue.(cause sometimes I got the value 3.4ug/ul but on gel it was hardly visible!!!)

Thanks for all replies
I read and pasted here what I found in the "Bible " of Molecular Cloning:

Measure concentration of DNA:
It is often difficult to measure the concentration of high-molecular weight DNA by standard methods such as absorbance at 260 nm. This is because the DNA solution is nonhomogeneous and usually so viscous that it is impossible to withdraw a representative sample for analysis. These problems can be minimized by withdrawing a large sample (10-20 ul) with an automatic pipetter equipped with cut-off yellow tip. The sample is then diluted with approximately 0.5 ml of TE (pH 8.0) and vortexed vigorously for 1-2 min. the absorbance of the diluted sample can then be read at 260, 270 and 280 nm.
(Adapted from the Molecular Cloning laboratory manual from Sambrook)