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18
by 50%, thus increasing the average extension length of each primer before a
ddNTP is incorporated. Conversely, adding 1µl of [α-
33
P]ddNTP will decrease
the ratio by 50%, thus decreasing the average extension length of each primer.
Running sequencing gels which resolve more than 600 nucleotides requires
high quality apparatus, chemicals and attention to many details. While specific
instructions are beyond the scope of this manual, following are some general
guidelines: The gel should be loaded with 8 adjacent lanes (GATCGTAC or see
‘Supplementary Information, denaturing gel electrophoresis’ section) with a
sharkstooth comb and be run 4 to 10 times longer than usual. For this kind of
experiment, gradient (or ‘wedge’) gels or very long gels (80-100cm) are almost
a necessity. The highest resolution gels appear to be approximately 6-8%
acrylamide and are run relatively cool (40°C).
Denaturing gel electrophoresis
Under optimal gel electrophoresis conditions, 250-300 bases can be read from
the bottom of a standard size sequencing gel. The length of time the gel is run
will determine the region of sequence that is readable. Many factors can limit
the sequence information which can be determined in a single experiment.
Among these are the quality of reagents used, the polymerization, the
temperature of the gel during electrophoresis, and proper drying of the gel after
running. The greatest care should be given to the pouring and running of
sequencing gels. The specifics of running the electrophoresis will depend on
the apparatus used. The following suggestions for reagent compositions and
procedures are intended as guidelines. For specific instructions contact the
manufacturer of the gel apparatus used.
Gel electrophoresis reagents
This kit contains a prediluted enzyme mixture which contains a high glycerol
concentration, requiring the use of a glycerol tolerant gel buffer. The use of
other buffers such as TBE can result in severe distortion of sequencing bands
in the upper third of the gel. The following recipe is for typical sequencing gel
reagents.
Buffers
20X Glycerol Tolerant Gel Buffer (71949 or 75827)
Tris base 216g
Taurine 72g
Na
2
EDTA
.
2H
2
O4g
H
2
O to 1000ml, filter (may be autoclaved)
This buffer can be used with samples containing glycerol at any concentration
(20). If gels seem to run a bit slower with this buffer at 1X strength, use it more