Question re: DIG DNA detection and "Tie-Dye" problems....

[TIM CHIPMAN]

I have a question for all people who have sucessfully used the Boehringer 
Mannheim DIG DNA labeling and detection kit. I have gone through the 
procedure  a few times now. The first time doing the procedure, I used the 
control DNA that came with the kit, and it worked perfectly. However, in the 
4 attempts I have made since then, I have had varied degrees of sucess, 
ranging from partial failure to total failure.

First puzzle:
 
I did two membranes in parallel, both with the same solutions, temperature 
and duration washes, etc. One of the two membranes  came up a complete tie-
dye pattern.
In the other membrane, the control DNA lane lane "lit up", but all 
other lanes failed. The absence of other lanes lighting up might have been 
due to too low levels of DNA possibly? We used approx 5ul DNA solution 
loaded per well, with DNA at a conc. of (approx) 250 ng/ul. {aside: Does 
anyone have any comments on what levels of DNA they have used sucessfully?}

Second Puzzle: 

I repeated the procedure, using a dilution series of DNA of varied levels, 
to see if I could detect what level of DNA was required for detection. I 
loaded lanes with 20,15, and 10 ul of DNA suspension, again at levels no 
less than (approx) 250 ng/ul. (DNA extracted from one honey bee...). Went 
through the whole kaboodle, and it developed with the control lane DNA (M-
13 probe, m-13 DNA in the control lane) visible; no other lanes lit up; and 
the infamous tie-dye pattern. Blargh. 

I followed the standard protocol to the letter. I was particularily 
carefull on the  most recent attempt; as I am getting very tired of the 
pretty tie-dye pattern (as is my advisor here...).

Does anyone have any recommendations or comments? I realize  that the tie-
dye pattern is a classic symptom of poor (low and high) stringency washing. 

Thanks very much! I appreciate your time!

Tim Chipman
901106c at axe.acadiau.ca




........    Tim Chipman                ..................................
..........    901106c at axe.acadiau.ca       ..................................