A260/230 readings post gel extraction
Posted 13 August 2009 - 11:58 AM
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!
Posted 13 August 2009 - 06:15 PM
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.
Posted 12 March 2010 - 07:29 AM
by contamination by phenolate ion, thiocyanates, and other organic compounds.
thiocyanates like the guanidine thiocyanate in buffer QG.
Posted 12 March 2010 - 08:10 PM