agarose gel purification of cloning vector

newsnet customer customer at newsnet.com
Mon Jan 2 04:23:29 EST 2006


> I've been trying to prepare a cloning vector for use in preparation of
> genomic libraries.  I digested pBluescript II with BamHI, incubated
> with SAP and purified with the Qiagen QIAQuick kit. To test the vector
> I produced a ~500bp fragment by digesting a PCR product with Sau3AI.
> The digest was run on an agarose gel and the correct fragment was
> excised and purified from the agarose using the QBioGene Gene Clean
> kit.  Because I find that the Gene Clean kit always renders DNA that is
> unsuitable for UV absorption spectrophotometry (mysterious strong
> absorption at ~240nm) I cleaned the insert DNA again with the QiaQuick
> kit (after which I was able to quantify it reliably).

Shouldn't you be measuring absorption at 260nm?
I heard that some commercial kits make quantitation via spectrophotometry
unreliable but I don't know why.

> This vector/insert combination worked well (hundreds of white
> colonies), but I also saw a lot of blue colonies that I thought were
> probably due to uncut vector.  I'd added a 10-fold excess of BamHI, but
> I still could see what looked like a faint band of uncut, supercoiled
> vector an a check-gel.  So I prepared more digested vector, incubated
> it with SAP, and then ran it on a gel and excised only the band
> representing the linearized vector.  Again I used the Gene Clean kit
> followed by the QIAquick kit to purify the DNA from the agarose.  I
> expected the original high number of white colonies with a reduced
> number of blue colonies, but what I got was almost no colonies of any
> sort.  Just one or two tiny blue ones.  A repeat preparation of the
> vector gave the same result.
>
> It seems like somehow the gel extraction and purification process is
> ruining my vector.  And it seems to be specific to the vector side of
> things, since an identical process was used to prepare the insert and
> the insert works fine with non-gel-extracted vector.  I suppose I can
> learn to tolerate the high background of blue colonies, but the
> above-described result has aroused my curiosity and I'd kind of like to
> know what's going on.  Before undertaking experiments to investigate, I
> thought I'd solicit comments in this forum.  Has anyone else had this
> problem or does anyone have an idea what might be causing it?

Examining your gel under UV light for too long when your trying to excise
your bands can ruin your vector.

> All gels were prepared using 1X TAE and a garden variety agarose from
> Invitrogen, with 0.025X Cambrex GelStar added for UV visualization.
> Ligations were performed with T4 DNA ligase from Promega, overnight at
> 4C.  Cells were commercially prepared DH5a, chemically competent at
> >1x10e9 cfu/ug.

Make sure your using well established protocols.

Cheers,
Sharp Tool





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