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Miniprep DNA-strange nanodrop peak at 240-250nm?


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#1 tarquin

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Posted 31 July 2010 - 03:41 AM

I have just started a high throughput DNA miniprep procedure in our lab using Millipore plates (I adapted it for use in a plate centrifuge as opposed to a vacuum manifold).
However I don't use a lysate clearing plate, instead I pellet the debris and transfer the cleared lysate as best I can to the DNA binding plate.
After 2 washes with 80% Ethanol the plate is spin dried for 10 min. I then elute with water, or TE.

On both occasions (TE or water elute) my nanodrop readings were strange. There was a sharpish peak between 240-250nm. There was still signal at 260nm, although this was the downward slope of the ~245nm peak, not a separate 260nm peak. Obviously my 260/280 ratio was high (2.2-2.7).

I've search the net to no avail. I'm thinking ethanol contamination, cell component contamination, or even something from the filter plates (spin speed too high?).
Please help!

Many thanks for your time.

#2 bob1

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Posted 01 August 2010 - 01:50 PM

It could be fines off the column in the plate?

#3 mdfenko

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Posted 02 August 2010 - 06:33 AM

It could be fines off the column in the plate?


not likely, fines would show a broad range of absorbance.

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#4 tarquin

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Posted 04 August 2010 - 10:46 AM

It turned out to be the bind buffer (Potassium Iodide).
Thanks for your replies anyway.

#5 WeeYorkie

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Posted 10 November 2010 - 04:15 AM

Hi, just wondering how you came to this conclusion?

I've currently got a huge peak around 250 and there is sodium iodide in the kit i use? I've found it quite hard to identify what is causing this the main contenders are EtBr maybe agarose (possible but unlikely I was quite careful!)and now possibly the bind buffer :/

just wondering how you determined what it was :)




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