Summary of Methods related to use of electrolyte gradient for increasing
band resolution on sequencing gels.
Last month I posted a query about using wedge gels to compress bands on
a footprinting gel. I have received many replies both through the news
group and via Email, and I believe it would be beneficial to summarize.
There are three main approaches, all of which rely on creating an electric
field gradient in the gel, such that smaller fragments are retarded relative
to larger fragments.
I. Wedge gels: An electrical field gradient can be created by varying
the thickness of the gel, from top to bottom. The simplest means of doing this
is to pour the gel using commercially available spacers. Most commonly,
the spacers taper from 0.2mm at one end (proximal to the wells) to 0.4mm
at the lower buffer chamber end. The commercial source mentioned most
frequently in replies was IBI (Kodak). One respondent indicated that
such spacers can be made at an institutional machine shop: order 0.4mm (or
thicker) nylon plastic sheets. Cut into strips. Attach double-face tape
to one side to anchor the strip to a lathe, then have machine shop cut
the piece, sloping from 0.4 to 0.2mm. The tape is necessary to maintain the
position of the thin plastic on the cutting platform. Another method is to
overlap thin spacers, stacking them to increase thickness. This approach is
more likely to leak, so tape the edges. Two references providing methods and
theory for this technique:
Ansorge, W, and Labeit, S. 1984. Field Gradients improve resolution
on DNA sequencing gels. Journal of Biochemical and Biophysical
Olsson, A, Moks, T, Uhlen, M, Gaal, AB. 1984. Uniformly spaced
banding patterns in DNA sequencing gels by use of field-strength
gradient. Journal of Biochemical and Biophysical Methods 10:83-90.
II. Buffer Gradient Gels:
Gels can be poured with a true gradient in the acrylamide mix. The
procedure is described in both Sambrook et al. ("Maniatis", 13.47) and in
Current Protocols (7.6.7). This method relies on two solutions of different
ionic strength, used in pouring the gels. No wedge is used, only spacers of
uniform thickness. The effect is similar, though, since the ion gradient in
the gel has varing conductivity (or resistance). References:
Biggin, MD, Gibson, TJ, Hong, GF. 1983. Buffer gradient gels
and 35S label as an aid to rapd DNA sequence determination.
PNAS USA 80:3963-3965.
III. Electrolyte Gradient Gels:
This is a variation on the buffer gradient gel. Instead of pouring
the gel with an ion gradient, a normal gel is poured, using normal spacers.
After pre-running the gel as usual, a salt solution is added to the lower
buffer chamber. When power is applied, the salt migrates into the gel, and
again alters the conductivity at the lower end of the gel. According to the
responses received, this appears to be easiest and least expensive of the
methods. The basic technique and theory is found in:
Sheen, J and Seed, B. 1988. Electrolyte gradient gels for DNA
sequencing. Biotechniques 6(10):942-944.
This technique is summarized in Current Protocols (7.6.8). According
to the responses received, this technique appears to be favoured over the
wedge technique, with the buffer gradient technique coming in a distant third.
Several variations on the basic technique are used in various labs:
In the basic protocol, the upper buffer chamber is 0.5x and lower
is 1.0X TBE. The gel is 1.0x TBE. The salt is 3M sodium acetate
and is added to a final concentration of 1.0 M immediately before
sample is applied. Some of the variants are:
* 1.0X TBE in both upper and lower chamber
* gel composed of 0.5X TBE
* add NaOAc 1 to 2 hours after start of electrophoresis
* variable %T gels, between 4% and 8%.
These techniques result in a thermal gradient from top to bottom (from
cathode to anode) with the top being hotter. The consensus is to run the gel
at 65W, lowering the power if it appears the plates are in danger of cracking.
Several replies suggest aiming a fan at the upper buffer chamber to draw off
heat. The heat itself will not harm DNA, and can only assist in suppressing
secondary structure and maintaining single strandedness. Earlier posts
suggested that the thermal gradient was responsible for the altered migration
and consequent compression of lower size bands. This is not the case, as
all three techniques rely only on electrical considerations. There are,
however, other techniques which use thermal gradients for detection of
secondary structures or in protein gels, to determine thermal denaturation
Thank you to everyone who replied:
Anyone else who I missed in compiling this summary.
John J. Welch
Roswell Park Cancer Institute
Buffalo, NY 14215
welch at sc3101.med.buffalo.edu