A260/230 readings post gel extraction - (Jul/22/2009 )

So I have recently had trouble with cloning and decided to look at every step in depth to see where the problem might be, and I stumbled upon the 260/230 readings I've been getting from the nanodrop. I found that generally you shoot for a 260/230 ratio of 1.7+ and the inserts I've been using in my ligations have had ratios of ~.05, incredibly low. I've never even checked this in the past since I never had problems so I'm unsure of what ratio I got from successful ligations. Currently I'm using the Qiagen QIAquick Gel Extraction Kit and taking readings after purification with that.

Basically I was just wondering what types of readings other people get after gel extractions, and if this could have a huge impact on downstream applications such as the ligations I'm doing.

Thanks!

psb on Jul 22 2009, 04:09 PM said:

I could be mistaken, but I thought the 260/280 ratios were supposed to be >1.7, not the 260/230 ratios.

If the 260/280 is around 0.5, then I'd suspect that could have a large impact on downstream applications. For the kit, make sure you add the 500 ul of buffer QG (I think that's the one... the one you melt the gel in). I'm not sure if there's a recommendation on that buffer to incubate 2-5 minutes, but if there is, do it. That 2-5 min may be for the buffer PE, though. Either way, for any of the "optional" steps for the qiaquick kit, do them. Usually the kit will give a poor yield, but the quality of the prep usually isn't that bad unless one of the steps is missed.

fishdoc on Jul 22 2009, 02:25 PM said:

The nanodrop support says to expect a A260/230 ratio of ~2.0:

Here's where I got that from:
http://www.google.com/url?sa=t&source=...89kE3zj8pOlWHpQ

I believe for 260/280 ratios it should be >1.8, though I might be off on that.

fishdoc on Jul 22 2009, 02:25 PM said:

I've done all the optional steps and still no luck! I was thinking part of the problem is that 95% ethanol was added to the wash buffer instead of 100% ethanol, however it was not denatured ethanol so it should ok I believe, do you have any thoughts on that?

psb on Jul 22 2009, 04:36 PM said:

I think the 95% would be fine.

Is there any chance the gel wasn't completely dissolved when you added the sample to the column?

I've used this kit frequently, and my only issue has been with yield that I can remember, so I'm not sure what could cause that much contamination other than maybe some agarose getting through to your sample.

I use the qiagen gel extraction kit all the time and have never encountered such a low 260/230. Regardless of 260/230 or 260/280, it is always optimal to get at least 1.8, though 1.7 is fine.

I always carry out the gel extraction optional steps and they have always gone fine. Have you tried using another set of qiagen gel extraction kit other than the ones that you haves, like say... get a bit frm your colleague and try and see if it works?

One other thing... what were the concentrations of your samples you ran on the nanodrop? I've had situations where the ratios were way off, but it was because there was such a low level of DNA present that the spec readings were really low, which then threw the ratios out of whack.

jiajia1987 on Jul 22 2009, 06:35 PM said:

We have a new kit on the way so we will see if that's the issue!

fishdoc on Jul 22 2009, 06:41 PM said:

The concentrations are ~20ng/uL, would this be considered low?

psb on Jul 23 2009, 10:39 AM said:

I think 20ng/uL is considered a little low, but it isn't too low either. It should still be enough for your work.

How did you prepare your inserts? After preparing your inserts, you got around 20ng/ul and the low 260/230 reading?

jiajia1987 on Jul 22 2009, 07:52 PM said:

The insert is PCRed from chromosomal E. Coli template DNA. After PCR purification (Qiagen kit) the entire volume (eluted in 30 uL) is double-digested. Following that digest it is run on a gel and then gel extracted(Qiagen kit)--although now that I think about it we might as well just PCR purify again since the ends we're cutting off are ~8bp or so. After gel extraction/gel purification we take the concentration reading.