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A260/230 readings post gel extraction


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19 replies to this topic

#16 Roo

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Posted 13 August 2009 - 11:58 AM

I had this problem off and on for quite some time. As it turns out, it was the columns in my Qiagen kit. I also thought the alchohol might be to blame, but borrowing fresh buffer with fresh ethanol didn't seem to help. I think our kit was just too old since the problem went away upon receiving a new one.

#17 jiajia1987

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Posted 13 August 2009 - 05:05 PM

I had this problem off and on for quite some time. As it turns out, it was the columns in my Qiagen kit. I also thought the alchohol might be to blame, but borrowing fresh buffer with fresh ethanol didn't seem to help. I think our kit was just too old since the problem went away upon receiving a new one.



great to hear that!

#18 fishdoc

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Posted 13 August 2009 - 06:15 PM

Since this topic came up, I've paid more attention to my 260/230 ratios for DNA preps. The gel purifications are pathetically low. I think I generally have less than 1.0 (probably less than 0.5) most of the time for gel purifications. That's with a Qiagen kit that's not too too old. A few months probably. It hasn't affected any downstream applications that I've noticed. I've used the DNA for successful cloning, anyway. Can't think of any time I've used it for sequencing, maybe it would have an effect there.

The 260/280 values are always >1.75, and usually always >1.8.

For comparison, PCR purifications and plasmid preps give the same high 260/280, but also have a 260/230 comparable to that of th 260/280. On rare occasions they're down around 1-1.5, but for the most part pretty high.

I've let the PE buffer sit on the column for up to 5 minutes before spinning, but that apparently doesn't do much.


Like I said, this hasn't caused me any known problems, just an observation about the spec readings following gel purification.


Another guy in the lab had some RNA samples he ran on the nanodrop and the 260/230 ratios were pretty poor. He called Qiagen (RNeasy kit) to find out if the low ratios would give a poor result for qPCR. They said the absorbance there was likely due to salts in the sample (which is what the EtOH wash in both kits is supposed to remove), but wouldn't have an effect on qPCR. Not sure if that's accurate or they were just giving a quick answer, but maybe excess salts cause the same problem for gel purification samples.

#19 Sudders

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Posted 12 March 2010 - 07:29 AM

Just thought you guys might like to know that according to wikipedia (that font of all knowledge) that absorption at 230 is caused:

by contamination by phenolate ion, thiocyanates, and other organic compounds.

link

thiocyanates like the guanidine thiocyanate in buffer QG.

Ian
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#20 phage434

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Posted 12 March 2010 - 08:10 PM

I wash twice, and make sure some of the wash lands on the inner ledge of the column, which I think can trap liquid. This seems to help a good deal with A230 problems.




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