Dear All,
I have been getting a problem during gel DNA recovery. When I checked the concentration of the DNA, purified from Gel, in the Nano drop, it is giving me 27 ng/uL, but the peak is showing at 235Nnm. In addition, the ratio of A260/280 is 2.1o which is very much acceptable what I have understood so far. Why is DNA giving the peak at 235 instead of 260? The A280/260 is 2.14.
Hi Abir,
looks like residuals from the gele to me. Check the ratio of 260/230. It should be over 1.8 when i'm not mistaken. Lower values indicate contaminants.
Bet regards
Hi there,
High absorbance at 230nm is due to traces of guanidinium coming from the purification buffer. An extra wash should lower the level of absorbance. The presence of guanidinium has no effect on downstream application. BTW to discuss recovery yield it is better to give the total amount of the DNA of interest loaded onto the gel and the amount recovered rather than the concentration.
Hi,
I agree with the above advice, buffer components can absorb at the 230 nm and the best control is to run a sample of the purification buffer alone, in case you used pure water as a blank. In case you used the purification buffer as a blank, you should get rid of the most of the peak at 235nm.
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