Problems with qiaquick gel purification kit - cloning (Dec/01/2010 )

Hello all:

I need some help with my gel purification procedure. I'm using Qiagen kit (Qiaquick) and i'm having extreamly low DNA amounts.
I'm cutting close to 2.5ug of vector (6.5kb) and i'm loading it into a 1% agarose gel. Then i cut the strand (6.5kb) as close as i can, and then i follow the protocol step by step (3vol. of QG buffer, heat during 10' at 50C - i'm checking the complete gel dissolution and the pH seems to be right- then, i add 1vol of isopropanol- mix with the pipet-, then pass it throught the column, centrifugate 1min at 13000rpm, add 500ul QG and centrifugate, then PE wash - i let the column with PE buffer for 5 min before centrifugate-, and finally elution in 30 ul -letting the column with the EB for 2 min before centrifugate.)
The last time i did it, i get 13.5ng/ul in 30ul, with ratio 1.80 260/280 and ratio 260/230 0.5. As you can see I'm getting very low amounts of DNA and the most important thing, my 260/230 is awful!
I'm already having nightmares with this protocol and my labmate continue to telling me "you have to be more carefull" but i don't know which step is the one i'm doing wrong.
Thanks for your help,

Losybelle

Gel extraction is never efficient. If you are just cutting the vector for cloning and not cutting out an insert of more than about 50 bp, you can just extract the plasmid with a PCR clean up kit which are designed to get rid of short DNA sequences.

bob1 on Wed Dec 1 22:29:55 2010 said:

You should make absolutely sure that you have spun the column dry before eluting. Alcohol in the PE wash which is left on the column can prevent elution. Another approach (recommended) is to elute a second time with EB. This will guarantee that the second elution has smaller amounts of EtOH.

But I agree -- gel elution is the last thing you want to do. You should design your cloning approaches to avoid it as much as possible, in my opinion.

phage434 on Thu Dec 2 00:19:57 2010 said:

You can also add the eluate back onto the column and re-elute, which usually increases the yield.

I use the same kit frequently and have the same problems as you every time. I feel lucky to get 30% of my starting material back, and the 260/230 ratios are always extremely low. It's the kit, not you.

I use the DNA for cloning all the time, without much problem. I've gel purified both vector and insert DNA for cloning. I've also used the gel-purified product for direct sequencing, which also worked fine.

It's not the kit -- I've gel purified every fragment of every cloning I've done in the last 10 or so years using the Qiagen kits, as have all the post-docs that have come through the lab during that period, as well as all the summer students, visiting scientists, etc. It works very well in our hands -- I honestly can't recall a cloning experiment performed in our lab in which the fragments were not gel purified, nor a cloning experiment that didn't give us the clone we were looking for by, say, the third attempt -- the vast majority work on the first try. BTW, we also never bother to get any spec readings because we don't have a Nanodrop (it's in another lab down the hall), and because it obviously isn't necessary -- we will usually eye-ball the ratios from the band intensity of the EtBr-stained gel from which the fragments are recovered, but that's it.

What we do that is different from most is to add guanosine to the gel and the TAE in which it's run (~1 mM, see here) and we use Ready-to-Go T4 ligase.

So, it's either not the kit, or we've had a 10-year run of incredible luck...

to summarize,

You are loosing DNA during the extraction process for two reason
1 - DNA is not binding to column matrix
2 - DNA is not dissociating from the matrix

You can improve situation (1) by
A - increase the amount of QG buffer use to dissolve the gel plug. More QG buffer, means better binding to the column.
B - pass the dissolved QG gel -DNA wash solution through the column several times
(3 times) to give the DNA more opportunity to bind
C- add isopropanol, at volume of 100ul isopropanol for every 300ul of QG-DNA solution.

Situation (2) can be improved by
A - using preheated EB elution buffer (about 68C) and leaving the EB buffer on the column for 1 minute to equilibrate (if possible at 68C) before spinning down.
B- if you are eluting the DNA in 50ul volume, do so into 2 steps. The first step is an elution of 20ul. The second elution is 30ul.

Lastly, cut more DNA. and saturate the column. You are going to lose DNA when using a column) so cut enough DNA to compensate.

The other thing that might be throwing you off is your initial spec reading. A high spec reading initially is leading you to conclude that you're starting with a ton of DNA (2.5 ug) when in fact the spec is telling you you're starting with a ton of stuff that absorbs at 260 nm -- including organic compounds, chromosomal DNA, etc.

How are you doing your plasmid extraction? Does the band you're cutting out really look like 2.5 ug when on the gel?

Your final concentration might be closer to the truth -- assuming you recover 80%, perhaps you're really loading only 17 ng/ul...

Why are you using a 1% gel?

Can you back up and give us your whole procedure? Have you tried cloning using your recovered vector, regardless of what the spec is saying?