Carried out a X5 & X10 dilution and when using the equation: Concentration (ug/ml) = A260 x dilution factor x 50 ug/ml, I did not get the same concentration for both. should I have had the same concentration for both?? and from my dilutions how do I find the amount of DNA in my total sample??
My suggestion would be to carry out an agarose gel electrohoresis and find the relative concentration of the DNA by comparing it with the standard ladder because during absorbance it is possible for both DNA and RNA to absorb at 260 nm and hence you will have higher concentration than actually present in the solution. However, as to your confusion regarding the different concentration in different dilutions, it could simply be due to crowding effect i.e. in lower dilutions you could have more aggregates of RNA giving the misread while in higher dilutions they get more dispersed and again give you the misread. Hence, agarose gel electrophoresis would be the best fit, or you can treat the sample with RNase and again perform the reading, however, this too doesn't give you a much reliable result although better than earlier. Usually, the absorbance reading comes to be accurate when you are using a high grade DNA extraction kit because in that case, most of the RNA and proteins can be removed if the experiment has been performed correctly.
Hi there,
In theory you should get the same result but experimental error occurs (pipeting, reading). How different are your results? Are they coherent or not? What are the reading you get? Do you read at 280nm too? 260/280 ratio is important for judging sample quality (should be between 1.8 and 2 for DNA)
As mentioned above reading at 260nm is specific of the bases contained in nucleotides but can also be affected by contamination from your extraction/purification method. If you want to be sure about your sample content go for agarose gel too.
agree with @ Mitesh Shrestha
do an agarose gel electrohoresis and locate the relative centralization of the DNA by contrasting it and the standard step in light of the fact that amid absorbance it is feasible for both DNA and RNA to assimilate at 260 nm and henceforth you will have higher focus than really show in the arrangement. In any case, as to your disarray with respect to the diverse fixation in various weakenings, it could basically be because of swarming impact i.e. in bring down weakenings you could have more totals of RNA giving the misread while in higher weakenings they get more scattered and again give you the misread. Henceforth, agarose gel electrophoresis would be the best fit, or you can treat the example with RNase and again play out the perusing, in any case, this too doesn't give you a much dependable outcome albeit superior to prior. Ordinarily, the absorbance perusing comes to be exact when you are utilizing a high review DNA extraction pack on the grounds that all things considered, the majority of the RNA and proteins can be expelled if the investigation has been performed effectively.
regards
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