A260/230 readings post gel extraction - (Jul/22/2009 )
You can actually carry out PCR purification after the digestion since the ends are so short.
I usually clean up my PCR products and take their concentration before I digest them. Do you? As for my vector, I will just digest 5ug. Did you take the concentrations before your digestion?
I agree with jiajia1987. 260/230 ratio should be >1.8 optimally. The last gel extraction I did was poor also, but is generally the case. The readings for my last gel extraction were: yield: 4.2ng/ul 260/280: 1.5 260/230: 0.01
I think the only real way to gel extract anything is the freeze/thaw method using phenol.
Hi, I have also had a problem with this, but completely opposite, my 260/230 reading is usually very high, in fact my highest one was around 13.0. This suggests to me that there is a purity issue, which would affect tranfection - my ultimate goal. Does anyone have any suggestions on possible issues which could affect the purity?
sorry, I meant 260/280!
nyx on Aug 3 2009, 11:08 PM said:
13.0 for 260/280 is really really really high. Either you have a very high concentration of DNA or there is something wrong with the quantification. What was the concentration of your DNA?
And, sometimes, when I do quantification, the values can go to negative values for the concentration of DNA, which really shocked me because that usually doesn't happen. What I normally do is to clean the nanodrop and re-initialize and re-blank again and check the concentration of the DNA again. Sometimes, the previous user might not have cleaned it up properly or the nanodrop has some slight errors; it is a machine after all.
The optimal value should be >1.8 for the spectro values. But I did read somewhere before that having too high a value could mean that there is excess RNA in the samples..
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.
Roo on Aug 14 2009, 03:58 AM said:
great to hear that!
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.
Just thought you guys might like to know that according to wikipedia (that font of all knowledge) that absorption at 230 is caused:
thiocyanates like the guanidine thiocyanate in buffer QG.
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.