measuring DNA concentration in a microplate - without pathway correction (Jun/10/2008 )
Hi,
if NanoDrop is not available and amounts of DNA are really low, the microplate spec should be possible to use for DNA concentration measurement. The question is - how? Pathlength of the plate is a unknown, because light absorbing path length of a liquid sample is not fixed by the walls of a cuvette. In modern spectrophotometers, there is a pathway correction function (not sure I understand how it works though...).
Is anybody out there who is using a UV-plate reader to mmeasure absolute DNA conc. and what do you do about the pathlength?
Appreciate any input,
Elena
If you have low amounts of DNA, I'd not try to use simply the A260 value. Instead, try the Hoechst dye method. Here are some links you can start with:
http://www.thelabrat.com/protocols/DNA-RNA...titation9.shtml
http://www.biotek.com/products/tech_res_detail.php?id=39
http://www.springerlink.com/content/h23060601u6l7841/
Thanks, but this is not what I am after 
With any fluorescence method, estimates are tied to the standard and this is what I am trying to avoid.
With any fluorescence method, estimates are tied to the standard and this is what I am trying to avoid.which plate reader and software are you using? I'd strongly recomend you contact the rep, and they might be able to help you with that.
in my case, we have a BMG POLARstar Omega, and the software has a pathlenght correction option (I dont know the theory behind it either) http://www.bmglabtech.com/application-note...itation-168.cfm
Another option is to use a standard curve, but I guess if you are trying to avoid that for fluorescence, you dont want that for absorbance either.
POLARstar is good I am using its earlier versioon - Fluorostar Optima, which is also not bad, just without the pathlength correction 
Unfortunately, local reps were not very helpful, all they did was trying to explain that we need ...a machine with pathlength correction
I suspect however that all this correction does is calculating a height of the liquid in the well, which is not that difficult to do manually, and doesn't take into account the menisk. If this is true, I wonder how reliable the concentration estimates and is there any caliblation studies between the cuvette spec and a plate reader...there must be... 
I am using its earlier versioon - Fluorostar Optima, which is also not bad, just without the pathlength correction Unfortunately, local reps were not very helpful, all they did was trying to explain that we need ...a machine with pathlength correction
I suspect however that all this correction does is calculating a height of the liquid in the well, which is not that difficult to do manually, and doesn't take into account the menisk. If this is true, I wonder how reliable the concentration estimates and is there any caliblation studies between the cuvette spec and a plate reader...there must be...
Not sure about correlation experiments, the only thing I can say (because I've done it myself) is the higher the volume you use, the most accurate the measurement. I've just OD some RNA and about to compare it with spec measurement, so will let you know. I think the readings on the POLARstar are quite skewed because I dont want to dilute my sample, and there's quite a lot of intereference from the buffer.
Yes, if I had enough DNA, I wouldn't bother measuring it on a plate reader, but this is not the case... And what I mean saying "calibration" is comparison between cuvette readings (1 cm pathlength, 1 ml and 2 ml, for example) and plate well (1 cm pathlength - 350 ul of the standard well). Would be very interested to look at your RNA OD. 