Abnormal Nanodrop Results for pTurbo EcoRI digest - (May/05/2009 )

I have been working on a cloning project for several months and dealing with multiple issues. I had thought I had everything resolved and decided to make new vector and insert to restart the process from the beginning. I did a single digest using EcoRI on pTurboGFP and pUC19. I then gel purified both using the Qiagen QIAquick Gel Extraction Protocol. When I used the nanodrop to quantify them I got some curves that I don't understand. The pUC19 peaks at a wavelenght of 230nm w/ ~13.2 absorbance (10mm scale), 260/280=1.86. The pTurbo peaks at 240 nm at ~13 absorbance, 260/280=1.93.

If anyone knows what those peaks mean I'd love to know.

The next step is to do a digest with XbaI.

Thanks,
Liz

I suspect you have some agarose contamination of your samples, there may be some ethidium bromide in there too, that could give you peaks similar to those you are seeing.

very likely to be actually the guanidine thiocynate ( i hope i spelled that right) it can absorb at that 230 wavelength . there's a website that talks about it but i don't have ti with me right now. u can perhaps go google about it.
EtOH precipitation normally gets rid of this problem.

You do not need to care too much about the peak. There are some contamination in it. But do not worry. You should just need to go ahead with the other digestion. If you have done too many times of digestion and ligation, you will never care too much about the Nano anymore for DNA concentration, I think.

The best way to get rid of agarose contamination would be to do a direct cleaning of your digested plasmids via a column, or even better: Simply do an ethanol-acetate precipitation.
Since you are simply linearizing your plasmids and not cutting out a fragment you don't need to do a gel purification...

The purification kit I used was over a column. Not doing the gel purification would make more sense, I just don't think it occured to me not to do it for the first digest.

I have had those nice curves before, but this time the peak and curve itself is tall and narrow.

Regarding ethidium bromide contamination, we've been using the SYBR safe stain instead of the ethidium bromide, and find that it works fairly well.

I'll do the 2nd digest and see how it goes.

mastermi on May 6 2009, 06:33 AM said:

I would be doing both digests at the same time, avoiding purification and DNA loss and the chance of damaging DNA by UV exposure. Less handling is almost always better.

GEl extraction is always my greatest enemy of all time. sometimes, despite many washes i always get some carry over. and when i see the sample won't freeze at -20C it makes the situation worse. For some reason the salt carry over is far severe for gel extraction than for other purification . i always wonder why. Agarose not washed away 100%, stuck in column and trapped salt together with them is my bet.