How to identify lanes on large agarose gels?

[Tom Anderson]

On Mon, 6 Aug 2007, Michael Sullivan wrote:

> Why not include something bigger in size than you expect for your PCR
> products in every lane, e.g. linerarized plasmid.

I reckon you want the opposite - something smaller, to mark the bottom of
each lane. If there's irregularity in running, it'll be worse at the
bottom than at the top, so that's where you need the marker.

How about using a loading dye with bromophenol blue, then taking a picture
in white light (assuming your gel doc does that) before going to UV? The
positions of the wells will be evident, marking the top end of each lane,
and you should be able to make out the BrPhB at the bottom. The only
problem is that BrPhB diffuses sideways a lot, so you might not be able to
discriminate individual lanes.

In that case, you'd need a small bit of DNA as a marker. You could run a
PCR for some random thing ~ 500 bp big, and then just use that. To get
something pure, maybe do two steps - a round of normal PCR to make a bit,
gel-purify that, then do a big preparative PCR with tonnes of magnesium
and millions of cycles to make loads of it. You wouldn't need to worry
about mispriming in the second round that way, and you don't need to worry
about linearity or errors or whatever anyway.

Alternatively, if you want markers on the cheap, make your own. Get some
plasmid (can be anything - use whatever you have most of in the freezer),
do a huge-scale digest with a couple of enzymes (loads of substrate, a bit
of enzyme, and leave it in the water bath over the weekend), and use the
result. Run it once against a standardised marker to work out the
molecular masses of the fragments if you want.

tom

> On Aug 6, 2007, at 1:09 PM, Peter Frank wrote:
>
> > I ran a large agarose gels for analyzing PCR products. The gel has
> > many narrow lanes. Problem is that there isn't a band in every lane,
> > sometimes there are several empty lanes between bands, and this makes
> > it difficult to identify the lanes because the wells are not visible
> > on the gel picture. However, I need to be able to clearly identify the
> > lanes so that I know where there is a PCR product and where there is
> > none.
> >
> > Drawing equally sized lines to locate the lanes did not really work
> > well because of the imperfection of the gel or gel run.
> >
> > Can you give me any tips on how to handle this problem? I was thinking
> > of putting a marker lane onto the gel every 4 lanes so that there
> > wouldn't be large spaces without anything. But I am not sure if this
> > is the best solution (besides, it would cost me a lot of DNA marker).

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson from ucl.ac.uk