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RNA Isolation


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#1 pauljb

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Posted 25 August 2009 - 04:56 AM

Hi All,

First post and time on one of these forums so please patient and i hope you can help.

I have human tracheal epithelial cell lines, i grow up on 24 well plates, take off and use Qiagen RNasy kit to produced pure RNA.

I have done this a couple of times now but still getting low 260/230 value. Everything else seems fine.

I have added in an extra step of buffer RPE and also i have added in a step of leaving the caps open on columns for 5 min to evap off any EtOH and also tried heating up RNA free water for last step.

These suggestions i got from google and did help with 260/230 value which is now up to around 1.5 - 1.8 but this is still a bit low. DO u have any suggestions for this?

Also i am not sure what the graph should look like on the nanodrop for pure RNA.

I think it should look this.

http://www.rr-resear...op_kurveRNA.jpg


However my graphs sometimes dont dip at 230 nm, more at 240nm (i still have a peak at 260) is this a problem? is this due to my 260/230 ratio being low?

Sometimes i get a peak at 230 and nothing at 260 what does this graph tell me?

Thanks for any help, i would like to be able to read more into the graphs, as i know what all the ratios mean.

Thanks again

Paul

#2 bob1

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Posted 25 August 2009 - 04:43 PM

Welcome to the forum.

low 260:230 is due to precipitated salts and possibly phenolics. I suspect that the problem is with the Qiagen kit, I have seen other similar posts from users of this kit. Try dissolving the RNA in 10 mM Tris pH 7.5, precipitate with isopropanol and re-suspend in tris again.

#3 pauljb

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Posted 26 August 2009 - 12:52 AM

Thanks,

I will give this a try.

Also have you any advice on reading the graphs produced?

i.e. for RNA isolation is it good to have a dip at 230 and a peak at 260? if so what do these dips and peaks mean?

Thanks again

Paul




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