Hello Jeff,

I suppose that you mean the 230/260 ratio, not 260/230, or else it means that there is something wrong with your Nanodrop. If that's the case, it means that you have got something that absorbs like crazy at 230 nm. I am not familiar with the PureLink kit, but there may be something in it (e.g. thiocyanate) that ma absorb at 230 nm. As to your negative minipreps, my guess is that what you detect with the Nanodrop is RNA. RNA absorbance at 260 nm is even higher than that of DNA, but it stains very poorly with intercalating fluorescent dyes like ethidium bormide

No, it's 260/230. Not 230/260. Besides, my scan shows much higher absorbance at 260 than at 230, hence they high number. If the ratio was 230/260 it would have been a very low number.

I thought about it being RNA. I did add the RNase while doing the minipreps but maybe it is off for some reason. The problem is, if that is true, why did one prep give me a band and not the other 5? They all had high 260/230 ratios.

Hi there,

Did you run the samples prior digest too? It could help you to check at what stage is the problem. The presence of a contaminating Dnase would be enough to give you such results but could not explain that your control is OK especially if you prepared the whole with the same kit and reagents

Thanks Dominique,

I ran the first miniprep uncut. That is the first lane, to the left of the first ladder. I did not run the other 5 uncut. I suppose it's possible the other 5 are there but something happened during the digest. It's weird that the one plasmid would be ok. I will try running all of them uncut, but that will be difficult since I am just about out of DNA. :(

I have now ETOH ppt the plasmids and the 260/230 ratios are much more normal but I still see no DNA on the gel.

You might not have done final spin well. some chemicals such as ethanol in column may have eluded into the final DNA sample.