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4 Notes
- The amount of enzyme used is crucial for the success of the
experiment and the activity of this enzyme varies between sup-
pliers. We generally start with 50 U of S1 enzyme in 6 mL of
1× S1 buffer and use 200 μL of this for each whole plug digest.
Once the gel is run UV illumination should reveal a bright
chromosomal band together with a faint smear of digested
chromosomal DNA throughout the length of the gel. If not
enough S1 enzyme is used there will be no smear and plasmids
are often not visible. If too much S1 is used the whole chromo-
some is digested leaving a smear of low molecular weight
DNA. The ideal amount of S1 should be determined empiri-
cally for each new batch of S1 enzyme by using a range of dif-
ferent concentrations of S1 from between 300 U per plug to
10 U per plug and a digest of 45 min at 37 °C. - Each standard PFGE gel has 15 wells, which is enough for 14
sample bacterial strains and a molecular standard ladder.
Washes and digestion of plugs for one gel or more are conve-
niently done in a 48 well culture plate. - Agarose gel is prepared by microwaving the solution for 2 min
and swirling the flask to see if any agarose crystals are not yet
dissolved. If agarose crystals are seen the agarose is re micro-
waved for 10–20 s time spans until no agarose crystals are visu-
alized. The hot agarose is then poured into the mold being
careful to remove all bubbles. - There is no need to stop the S1 reaction other than removing
the plug from the digest as long as the plugs are loaded and
run immediately after the digest. Ideally the plugs should be
loaded and the electrophoresis run started within 30 min as
longer periods will allow the S1 to degrade the chromo-
somal DNA. - The PFGE equipment is designed to run one gel at a time.
However, in practice we often run duplicate gels stacked on
top of each other. The advantage of this is that two identical
gels are prepared such that the same gels can be probed with
different probes. Most of the gel-running parameters are the
same when one or two gels are run but if two gels are run a
little extra running buffer will need to be added to the PFGE
tank to ensure that both gels are under running buffer. We
also add an additional hour to the run time when running
stacked gels. - When drying the PFGE gel between pieces of blotting paper,
ensure that a weight is placed evenly covering the length and
breadth of the gel. This will ensure that the gel does not fold
up as it dries.
Mark A. Toleman