PFGE trouble>Hi there, > My problem with my PFGE apparatus is I am getting streaks instead of bands. I believe that this is related to the fact that my buffer is giving slightly lower mA readings (18 instead of 21; 15 instead of 18) than usual. I have re-made the buffer exactly the same way that I always do when I get high quality results. I sent the power source in for repair, as I did 6 months ago when the same thing happened, and they found the the readings were slightly HIGH and re-calibrated the power supply. Since I got the power supply back, I have run a 24 hour gel with standards in 0.5 X TBE that came out OK. The normal gel that I am running, with 10 times less EDTA hence, 0.5 X TBe, has resulted in a continuous smear. >I also ran a PFGE gel on another machine, in another lab, with THE SAME buffer from the THE SAME bottle, and it came out well. Thank you, in advance, for your help. nkh@sage.purdue.edu

[Vivian Miao]

In article <dunkles_group-2601961038000001 at botany40.btny.purdue.edu>,
dunkles_group at BTNY.purdue.edu (Dunkles group) wrote:

> My problem is that I am getting streaks instead of bands.  I believe that
> this is related to the fact that my buffer is giving slightly lower mA
> readings (18 instead of 21; 15 instead of 18) than usual.  


... (stuff sniped out)

(This may sound strange, but ...) have you tried cleaning out your entire
PFGE rig, tubing and all?  I had a streak problem once and in that case it
turned out that some fungus or other was living in the buffer
(electrically hot and all) I guess, and just basically degrading the DNA
in the sample.  I think I flushed the equipment really well with water,
and then with ethanol.  Someone suggested I use Na azide, but I think
that's pretty toxic stuff.  Anyway, the problem was solved after that.