Both the amount and the accuracy of the nucleotide sequence depend on the quality of the polyacrylamide gels used to display the radiolabeled DNA fragments. Under optimal conditions, between 300 and 400 nucleotides can be obtained from a denaturing polyacrylamide gel. However, by electrophoresing aliquots of the sequencing reaction for different lengths of time, it is possible to read up to 500 nucleotides of sequence from a single gel.
The concentration of acrylamide used to prepare the gel depends on the size of the DNA fragments that are to be analyzed (see section pageintro). Sequences lying between 25 and 400 nucleotides from the terminus of the primer can be read from gels containing 6% polyacrylamide.
A 0.4 mm thick, 8 M urea, polyacrylamide sequencing gel was used. The glass
plates (34 
40 cm) were cleaned with a detergent and absolute alcohol.
The larger glass plate was layed flat on the bench and the two spacers arranged
in place along the sides. Then the smaller (notched) plate is layed in position,
resting on the spacers.
The plates were then clamped together using bulldog binder clips.
A buffer gradient gel was made to slow down short
fragments[125, 126]. The acrylamide solution
was prepared by dissolving 38g of acyrylamide and 2g of
N,N'-methylenebisacrylamide
in 100 ml water
at 37psy176 C.This solution was stored at 4psy176 C in dark. The Top
solution was composed of 25g urea, 7.5ml acrylamide solution, 2.5ml 10
TBE buffer and 21 ml of water. The Bottom solution was composed of 5g
urea, 1.5 ml acrylamide solution, 2.5m l 10 TBE buffer, 2.5 ml of water and
bromophenol blue
.
To seal the bottom of the gel, 5 ml top solution were polymerized with 15
l ammonium persulfate (25%) and 15 l TEMED . This fast hardening
gel was poured round the bottom of the plates and left for 20 minutes.
10 l ammonium persulfate solution (25%) and 15 l TEMED were added
to the bottom fraction, 50 l ammonium persulfate and 50 l TEMED
to the top fraction. They were mixed by swirling, and 11 ml of the
top solution taken up in a 25 ml pipette carefully followed by 6 ml of
the bottom solution. The solutions were allowed to gently mix by some air
bubbles going up through the pipette. The pipette was then emptied into the gel
plates. The remaining plate volume was filled by the top
solution. The gel has to be carefully examined for air bubbles. Bubbles in the
upper portion of the gel can sometimes be removed by knocking.
The mold was then layed down at an angle so that the top of the mold rested on a support about 5 cm high. This reduced the hydrostatic pressure at the base of the mold and prevented leaks and bowing of the gel plates. Immediately the flat side of a shark's tooth comb was inserted approximately 0.5 cm into the gel solution. The comb was clamped in position and the gel allowed to polymerize overnight.
The shark's comb was removed and the slot washed with buffer. The gel mold was
attached to the electrophoresis apparatus together with a metal plate to ensure
even diffusion of the heat produced during electrophoresis.
Approximately 500 ml 1 TBE were filled to the upper and lower buffer
reservoir. The shark's tooth comb was reinserted with its teeth just sticking
into the loading surface of the gel. The slots were washed with buffer once
again to remove any fragments of urea and polyacrylamide. The gel was loaded
with 2 l of loading solution to alternate wells. The gel was then run at 50
W constant power until the dye front ran a few centimeters into the
gel.
In the meantime, the sequencing reactions were heat-denatured (see section
heat) for 5 minutes and stored on ice until all samples have been loaded
onto the gel. 1.8 l of each sequencing reaction was loaded in A C G T order.
The gel was now run at constant power (40 W, 
1800 V) for the time
required to achieve optimal resolution of the sequence of interest (4 to 8
hours). The time required can be estimated by monitoring the migration of the
marker dyes in the formamide/EDTA/xylene cyanol/bromophenol blue gel-loading
buffer (see table tab:page).
Table 3.3: Migration rates of marker dyes through denaturing polyacrylamide
gels
At the end of the electrophoresis run, the gel mold was removed from the apparatus, and layed flat with the smaller plate uppermost. Using the end of a metal spatula, the plates of the mold were slowly pried apart. Now a piece of Whatman 3MM paper was placed on top of the gel. Applying gentle pressure the gel became firmly attached to the rough surface of the paper. Now the paper was slowly peeled from the glass plate. The gel was covered with plastic wrap and placed in a vacuum gel dryer. It was dried under vacuum at 80psy176 C for 2 hours.
Afterwards, the gel was removed from the dryer and the wrap peeled off. An autoradiograph was established by exposing the gel to X-ray film (Kodak XAR-5) at -80psy176 C for one to several days. The autoradiograph was developed and the sequence read.
Reading sequences from gels is not an easy task and skill is required. All gels were read and recorded at least twice. Discrepancies were resolved by further sequencing.