pulsed field gel electrophoresis

Tom Zahrt t-zahrt at uiuc.edu
Sat Dec 3 11:59:31 EST 1994


I am having problems in resolving DNA fragments when running the PFG. 
These problems do not appear to be due to the quality of the chromosomal
preps since yeast markers and low molecular weight markers purchased as
standards also resolve poorly.  The PFG's are run on a Beckman TAFE
Geneline system.  All of the runs have been at a constant
current-initially at 170mA for 30 minutes with switch times of 4s and then
150mA for 18 hours at a switch time specific for the size of the DNA
fragments I am looking for. I have been using Beckman LE pulse-field grade
agarose at a concentration of 1%, and running in 1X TAFE buffer (which is
approximately the same as .4X TAE).  The temperature at which the gels are
run is app. 12-14 C.  I have tried to correct the problem by trying a
number of different things.

1.  Changing the switch times does help distinguish between the larger and
smaller fragments, but the resolution of the bands seen is still poor. 
The standards seem to be fuzzy and not as clear as expected.

2.  I have tried to use ultrpure water instead of single deionized water,
but this didn't make a difference. In addition, different types of agarose
show the same result.

3.  The buffer in the gel box is changed after each run and fresh buffer
made immediately before each use, so I don't think that it is a problem
with buffer breakdown.

4.  I have tried changing the amount of DNA added to each well (cutting
the plugs in halves and quarters) thinking that I may be over or
underloading the lanes.  This has helped a little but the bands are still
fuzzy and not sharp as I would expect.


Any ideas as to what the problem may be??  Thanks.

-- 
Steve 
steve_gort at qms1.life.uiuc.edu



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