I want to calculate the concentration of DNA in an unknown sample. But it's buffer has a high concentration and our nanodrop don't accept that as a blank of dsDNA. Thus I dilute the buffer with distilled water in a 1:2 ratio and then there was no problem.
Now should I simply dilute my sample in the same ratio and check the absorbance? (is there a linear correlation?)
Unless the buffer is something that absorbs in the UV range, then the buffer concentration should not really matter. If you blank the Nanodrop against water and then measure the profile of just the buffer solution, do you get high readings or is it flat across the range.
The reason this is important is that if you have something in your buffer that absorbs in the UV range and it is at a high concentration (and high absorbance) then it will not be very accurate to just blank the machine against the buffer.
Nanodrops are generally unreliable. Try using a Qubit with 2 standard concentrations or alternatively you can try running it on an agarose gel against a product of known concentration and use the differences in intensity (ImageJ) to estimate relative concentrations. Hope that helps.
- Badges
- Science method