hi all
regarding DNA concentration determination by nanodrop , i just read that ((For reliable spectrophotometric DNA quantification A260 readings should lie between 0.1 and 1.0. ))
does that mean that any reading above this range is not acceptable ?
because I have high DNA concentration samples i.e 300ng/ul and I have the A260 is 3 and 4
so can i accept this reading ?
as for the criteria this is not acceptable. It will interfere in the process of PCR, so that the range should be at the standard value.
Set up a series of dilutions and measure OD, then plot the results. You will see that at some point between OD 1 and 2 the slope begins to plateau, so simply using an extinction coefficient for absorbances that high will underestimate the actual concentration of the samples. You need to dilute the samples if you want to get reliable readings.
Soma Yousef ,if PCR is running well, you got the desirable bands on the gel, i think it may be a great sound, but may have consequences in case of gene transformation.
The answer by Alejandro Martin is correct that you need to dilute your sample so that the reading is between 0.1 and 1 otherwise you are using the instrument in a non-linear range and you are not getting an accurate answer. So if you want an proper measurement of how much DNA you have, that would be the approach. On the other hand if all you are doing is using it as template for PCR, then you don't really need to worry. PCR is pretty tolerant over a wide range of template concentrations, so it just may not matter if the concentration you have is incorrect by 2-5 fold.
Dear Soma, I would like you to know here few tips and suggestions for the situation you have encountered.
In general, we must adhere to the standard acceptable ranges (like 0.1-1.0) when quantifying nucleic acids through a spectrophotometer, as the OD values, which we get from our samples are not necessarily reflecting the actual amount of our desired nucleic acid. It mainly also depends on our extraction technique and its accuracy.
Since lots of other but extraction-related substances may also absorb under UV spectrum, so just relying upon OD readings is in fact not really accurate for nucleic acids quantification.
It has been in common notice that whatever values we are getting through spectrophotometer or even the Nanodrop (ND), the actual concentration of DNA or RNA is always less than it shows.
For example; there are many substances which can also cause an increase in the OD260 readings like GITC, residual phenol content after the extraction may contribute to higher 260 values. Often, if the sample is contaminated with certain proteins rich in phenylalanine (F) during the extraction process you may get higher 260 ODs, as phenylalanine is known to have the absorption preference at 240-265 nm UV range.
So here your skills and technique for extraction does matter. That is why fluorescent-based approaches (PicoGreen, OliGreen) to quantify nucleic acids (DNA) are far superior and less error prone than the simple UV-based methodologies.
Regards....
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