Qiagen, Bane of My Existence

M

MaduroUTMB

2,500+ Posts
And so it comes to pass that I am exiled by the four winds to Austin (actually, one wind that moved in huge circle). I have chosen to pick back up on some unfinished lab work here, and I have come to realize that a lot of my headaches in the past were due to Qiagen.

Qiagen makes a lot of different molecular biology reagents, tools, you name it. However, at least for DNA purification kits, they don't make them WELL. I was putting 5 micrograms of DNA into a restriction digest and Klenow fragment blunt and tonight I got 1 back out. I used their stuff (not our water) and actually pondered blowing on the column to see if I could evaporate more of the ethanol wash buffer out (not that there is any left in there).

This did not help. Looking online (not on their website), I found that the Qiaquick column recovers DNA like Lehman Brothers recover mortage payments from SoCal. So now I get to heat the elution buffer to 60 degrees C with the goal of maybe getting a 50% yield. I have heard that invitrogen stuff is better. Anyone have any experience with that?
 
I've never had much luck using Qiagen preps either. My lab switched to Promega's kits (http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_1671) which I think are easier to use and give much higher yields.
 
I find all the columns pretty similar. Machery Nagel/Clontech, Qiagen, Invitrogen... What type of cells are you working with, what size fragments are you purifying, and how viscous is the digest? And I forgot what the maximum prep size is for the columns. Overloading the column is a common issue. Follow the directions closely without skipping steps and use fresh reagents for now.
 
It's digested plasmid DNA in a 3.5kb linear fragment. I got 3.2ng/ul the other day and I going to try to transform E. coli on Monday (shuttle vector FTW).

As for the steps, I'm following the longest and most tedious protocol that they offer. If I have to do these purifications again, I will probably heat the elution buffer to 55 or 60 degrees C.

Edit: I wind up doing one purification from a digestion and one from a gel. In both cases I got 15-20%.
 
That yield is a little low. Is the 260/280 good? If not, you will probably have some problems with the transformation. Purifying by gel extraction, I assume you are cutting somewhere around 400 - 1500bp. I also assume that you performed a serial digest and are getting a good digest without a lot of uncut DNA when doing the prep procedure. As you know the prep procedure can vary a little from the serial digest.

If the band is pretty large, separate it into 2 gel extraction columns so as to not clog the column. Still do the extra wash to get better purity: It won't effect yield that much. Also, double check that you added alcohol to the PE reagent bottle of the QIAquick Gel Extraction kit. Elute from one of the columns as directed and then use that eluate to elute the DNA from the second column. I would assume in the end that you would get much, much better yield than the 3ng/ul.

Hopefully, you have something like a nanodrop so as to not use too much of your DNA for analysis. Good luck and if you need further help beyond what Qiagen can provide and are totally stuck and at a dead end, email me with the complete info. of what you are doing.
 
The starting material is purified plasmid DNA. Qiaprep works great for us.
Anyhow, I found the problem. The 260/280 ratio is 2.00, but the 260/230 ratio is 0.05. That's not a typo, and that's after spinning the PE buffer out for an extra 3 minutes at 13k RPM (meaning that I spun the buffer out, emptied the catch tube, spun it for 1 minute, checked and saw nothing there and finally spun it again for 3 minutes just to be sure).

I would be sure that the elution buffer somehow has EtOH or salt or phenol or something unwelcome in it, but we got similar results with water (I assume that the poor yields have been due to residual ethanol the whole time). So now I am stuck trying to figure out how to get the PE buffer out, since it seems to be tenaciously holding on. Did I mention that I switched to a clean catch tube after spinning the PE buffer out the first time?

I may try centrifuging the PE buffer out at 13k for 5-7 minutes after I do the steps that Qiagen recommends (1 minute, empty, 1 minute).
 
I'm glad that you caught the problem, because I woke up this morning and realized that 250mgs isn't bad. When it gets up to the 400mg range is when you have problems.

What you mentioned is another common issue. In the manual it gives some possible solutions. Switching to a fresh tube is one of the solutions as is doing an extra spin. Its good that you caught it because otherwise the transformation probably wouldn't have worked anyways. Hopefully, your sample isn't too scarce.
 
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