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).
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?
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