Smiles in agarose gels
Dr.Sailesh Surapureddi
>Hello everyone.
>
> In order to separate DNA fragments obtained from RAPD, I make the
>amplification samples run through an horizontal agarose gel.
>Unfortunately, I always get this "smile" that bends all the bands and
>makes the gel analysis much more complicated. The gels are 1,5%, and I've
>tried different voltages from 20 to 80 volts, and although I noticed that
>the "smile" is reduced with decreasing voltages, it isn't clear enough to
>allow a perfect analysis. I'd be glad to hear any suggestion, advice or
>magic trick to improve the situation.
>
>Thanks in advance.
>
>Pierre-Alexandre Landry
>Dept. des Sciences Biologiques
>Universite de Montreal
>C.P. 6128, succursale centre ville
>Montreal, Quebec
>H3C 3J7
>
Hi Landry:
Atleast some body is smiling! Be gald for it.
Coming to your problem, smiling of the gels usually occurs with improper
castings, either the gel is being over or under boiled. I would check for the
quality of the buffer too, incase it got contaiminated.
A through wash of the gel cast also helps to a lot greater extent.
Smile as you sail away.
Sailesh.
Landry Pierre-Alexandre
WSc...@aol.com
Pierre-Alexandre) writes:
>Unfortunately, I always get this "smile" that bends all the bands and
>makes the gel analysis much more complicated. The gels are 1,5%, and I've
>tried different voltages from 20 to 80 volts, and although I noticed that
>the
If you aren't recirculating buffer, you might try this. Buffer does get
depleted during gel runs.
Check your electrodes--are they full width of your cell? short electrodes
could concentrte current in the center of lyour gel to give you a smile.
Walt Schick
PS the super cell of Hoefer (Pharmacia) has cooling as well as recirculation
and gives flat patterns for RFLP and RAPD, you might try to get a demo from
Hoefer distributor in canads. Owl and Jordan also offer nice recirculating
designs.
marty_carson
lan...@ERE.UMontreal.CA says...
>
>Hello everyone.
>
> In order to separate DNA fragments obtained from RAPD, I make
the
>amplification samples run through an horizontal agarose gel.
>makes the gel analysis much more complicated. The gels are 1,5%, and
I've
>tried different voltages from 20 to 80 volts, and although I noticed
that
to
>allow a perfect analysis. I'd be glad to hear any suggestion, advice
or
>magic trick to improve the situation.
>
>Thanks in advance.
>
>Pierre-Alexandre Landry
>Dept. des Sciences Biologiques
>Universite de Montreal
>C.P. 6128, succursale centre ville
>Montreal, Quebec
>H3C 3J7
small amount of EtBr to our ficoll loading dye (1µl EtBr stock
(10mg/ml) per 1ml ficoll). I'm assumming that each band within each
lane is 'smiling' and its not just one big smile across the whole gel.
Hope this helps.
Manuel G. CLAROS
> Unfortunately, I always get this "smile" that bends all the bands and
Try to run less DNA in every sample. Excess of DNA (as well as RNA) make
the bands smile :-)
I hope this unsmiles ;-)
--
Manuel G. CLAROS -. .-. .-. .-. . -> cla...@obelix.cica.es <-
Biologia Molecular ||X|||\ /|||X|||\ /| No quiero cambiar, no puedo,
Facultad Ciencias |/ \|||X|||/ \|||X|| no quiero ser una frase mas.
E-29071 Malaga ' `-' `-' `-' `- (Esclarecidos)
Susan Jane Hogarth
>Check your electrodes--are they full width of your cell? short electrodes
>could concentrte current in the center of lyour gel to give you a smile.
Is this true? A grad student in my lab runs starch (protein) gels with a piece
of platinium wire just clipped into the buffer compartment - no more than 1
inch long. He doesn't seem to get any radical side to side variation on the
gel. Is it different with agarose (DNA) gels?
--
Susan Jane Hogarth
http://www4.ncsu.edu/unity/users/s/sjhogart/public/home.html
WSc...@aol.com
Hogarth) writes:
>Is this true? A grad student in my lab runs starch (protein) gels with a
>piece
>of platinium wire just clipped into the buffer compartment - no more than 1
>inch long. He doesn't seem to get any radical side to side variation on the
>gel. Is it different with agarose (DNA) gels?
>--
While at biorad, we designed an agarose bridge cell for immuno
electrophoresis, and found that there was a voltage drop along the platinum
wire. Enough to affect the sample run, so we connected the anode and cathode
to the power supply at opposite corners of the cell.. This gave a uniform
electrical gradient. Most commercial DNA submarine and vertical protein
cells also follow this practice. The amount of buffer in each tank, and
its conductivity also change the field strength, and can affect gel patterns
during the run.
.
From field reports, the old IBI DNA cells gave good non smiling gels by
locating the electrodes UNDER the gel platform.
Running gels slower reduces the heat generated inside the gel and reduces
smiling effects. Most cells, even with pieces of platinum wire, will give
fair gels by running overnight. The best designed cells should give uniform
patterns even after fast runs.
Note that the effort to provide uniform heating is especially important in
sequencing gels, and that most designs do use opposite corner electrode
connections and full length electrodes.
The best agarose and polyacrylamide patterns come from vertical cells where
the gel is in a sandwich of glass with similar cooling conditions on both
sides of the gel.
Electrode position is important here also. Some precast gel boxes have V
configured bottom electrodes, and the gel band pattern is affected.
Again, running slow allows electrode byproducts to mix uniformly, allows heat
to dissipate, allows individual molecules to equilibrate, etc. Overcomes
non-ideal physical/electrical configurations. Published patterns aren't
always the ideal, but the information is usually obtainable even from smiling
gels. Don't throw away your gel box, but when you shop for a new one, check
the electrode configuration. Most suppliers will let you demo a unit before
buying.
Walt Schick
Mr SMNN Faruque
electric field by the samples.
Samples loaded into the wells have a high resistance, but normally they
do amount to a large barrier. If a substancial cross-section of the
electric field is blocked by the sample (ie if wells are filled to the
top, in a tank with only a small amount of buffer above the agarose,
particularly when wells are joined to form a large well) frowning occurs
(ie the centres of the bands are slower than the edges).
Now that I load my samples in 1x buffer (with 2.5% ficoll and some dye)
I never see band distortion.
Nadeem