problem in 2D-PFGE

[David Chiu]

I am trying to map the gemone of certain bacteria. I use the modified
2D-PFGE method, ie run 1st dimension, cut out bands and digest with another
enzyme and label and run.

I was able to get good result twice. Now, however, I have ran out of luck.
All I can get is either smear of about 50kb or no visible band at all! I am
sure the end labelling works fine because I always include a single digest
end label plug as marker. I have tried nearly everything I can think of.

Once, I have tried to avoid uv totally. another time I have heat the cut
out bands at 65C for an hour. I always wash the gel slices for several
times with TE.

What ran wrong!?
Please help me! 

Rgds,
David Chiu
(bcdavid at usthk.ust.hk)
Dept. Biochem.,HKU of Sci & Tech