Consistent subcloning from agarose (finally !)

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Thu Apr 22 13:03:41 EST 1993


In article 4418 of bionet.molbio.methds-reagnts Jim Graham
wrote about a consistent subcloning technique and some further
discussion resulted. Some of the comments included the avoidance
of glass-milk for larger DNA fragments and certain transilluminators.

I've clipped some of the text from various parts of the discussion:

> I've had good results with agarose concentrations from 1.5% to
> 0.65% and fragments ranging from 100 bp to 11,060 bp

> Excise the bands of interest on a LONG WAVE UV box. A standard medium
> UV bulb will inactivate your vector fragment.

> As far as visualizing the bands, be SURE you have a 360 nm peak transillum.
> Ours is an old UVProducts model. Next to it is a new mid-range (302 nm peak)
> model, also from UVP, that is very commonly seen in labs. This one gives
> sterile transformant plates every time.

Well Jim, this week I tried everything wrong and still got it to work.
I was trying to ligate two DNA fragments sliced from a TBE gel. One
EcoRI fragment containing a replication region was 7.7 kb and the other
was a 4.5 kb EcoRI fragment containing a kanamycin resistance gene.
I first exposed the EtBr stained gel to mid-range UV (300 nm peak) for about
20-30 seconds at high intensity in order to photograph the gel. We just
bought a new FotoDyne Foto/Prep I. Although I used one of those special glass
plates used for sequencing gels on an ABI model 373A DNA sequencer between
the gel and transilluminator, I don't think it blocked any UV. I then
excised the bands of interest under the "preparative" intensity which
exposed the DNA for an additional 5 minutes. I gene-cleaned them as per
my previous posting, ligated overnight, and transformed DH5a with an
expression time of 1 hour. The next day I had ~ 100 colonies on kan plates.
I screened 18 colonies by mini-prep - the exact number that fit in my
microfuge rotor ;-) Of the 18 colonies, 16 were the correct fragments
in orientation I, one was the correct fragments in orientation II, and
one was that odd ball clone we've all seen that doesn't look anything
like the one expected (but not of lower MW than those ligated).

This proved to me several things:

1. The mid-range UV of 300 nm peak did not inactivate my vector.
2. The glass-milk method worked fine for fragments > 5 kb eventhough
   it's not recommended.

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA



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