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A260/230 readings post gel extraction


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#1 psb

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Posted 22 July 2009 - 01:09 PM

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!

#2 fishdoc

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Posted 22 July 2009 - 01:25 PM

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!



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.

Edited by fishdoc, 22 July 2009 - 01:29 PM.


#3 psb

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Posted 22 July 2009 - 01:31 PM

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


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

The 260/230 values for “pure” nucleic acid are often higher than the
respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. If the ratio is appreciably lower than
expected, it may indicate the presence of contaminants which absorb at 230 nm.


Here's where I got that from:
http://www.google.co...89kE3zj8pOlWHpQ

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

#4 psb

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Posted 22 July 2009 - 01:36 PM

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.


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?

#5 fishdoc

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Posted 22 July 2009 - 03:13 PM

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?



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.

#6 jiajia1987

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Posted 22 July 2009 - 05:35 PM

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?

#7 fishdoc

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Posted 22 July 2009 - 05:41 PM

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.

#8 psb

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Posted 22 July 2009 - 06:39 PM

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?

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

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.


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

#9 jiajia1987

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Posted 22 July 2009 - 06:52 PM

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?

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

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.


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


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?

#10 psb

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Posted 22 July 2009 - 07:14 PM

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?

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.

#11 jiajia1987

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Posted 22 July 2009 - 07:32 PM

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?

#12 georgiadave

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Posted 23 July 2009 - 02:46 PM

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.

#13 nyx

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Posted 03 August 2009 - 07:03 AM

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?

#14 nyx

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Posted 03 August 2009 - 07:08 AM

sorry, I meant 260/280!

#15 jiajia1987

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Posted 03 August 2009 - 05:03 PM

sorry, I meant 260/280!



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..




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