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
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).
in order to stain the wells, you might add some BSA to your loading
buffer. Another choice would be a fluorescent ruler which you might
photograph together with the gel. That might make it easier to locate
the lanes. Why do you get such strong deviations that you can't re-
assign the bands to lanes. Too much salt?
Does BSA stain with ethidium bromide as DNA does? Or how would that
So far, I used a DNA ladder (ruler) on each side of the gel for
orientation purposes. This worked well for smaller gels but for larger
gels that obviously wasn't enough to make locating of the lanes easy
within the gel. I guess I would have to put some DNA ladder lanes
within the gel, too. Your suggestion with the fluorescent ruler sounds
good. Can you tell me where I can order such rulers?
Maybe. Or temperature gradients within the gel while it is being run.
Michael Sullivan <[Only registered users see links. ]> wrote:
Yes, that would probably work. Do you think that would impair the
samples somehow? If yes, how about adding that plasmid into each well
shortly before I stop the gel run so that the linearized plasmid would
only run by itself and only a small distance into the gel?
I don't see how it would have an impact on the bands you are
interested in. I would think just adding some linear plasmid to your
sample buffer so that you get a concentration of say 20 ng/lane would
On Aug 7, 1:11 am, Peter Frank <[Only registered users see links. ]> wrote:
I think with a little tweaking of settings on the camera/photodocum.
system (especially brightness and contrast, aperture and shutter) you
should be able to see the wells on the gel. If you only interested in
the yes/no result there is no need for rulers and ladders, only
positive control of expected size.
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 Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) [Only registered users see links. ]
"Peter Frank" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ]...
when a PCR gives you a band, you see it... when it doesn't, the primers
don't get used up and you *may* be able to see them, depending on how much
you use per reaction.
In cases like that, I just take two pictures of the gel, one to see the
bands nicely, and another tweaking exposure/brightness so that i can see the
wells, even if most bands appear overexposed... between the two I identify
every single lane.