More Spacing Questions

IHG Steve
Mon Apr 14 11:05:39 EST 1997


Lately I have been having trouble with out ABI373 machine being able to
space properly. Many of the lanes end up with the infamous -12. The
control has been hit-and-miss, although if I reanalyze I can usually get
it to space properly. When I look at the gel image it looks pretty good,
although the bands don't appear to be resolving perfectly--they look
somewhat smeared. When I look at the analyzed window the bands appear to
be spaced evenly and the signal strength is fine. This problem seems to
have come on just in the past couple of weeks. My client reactions appear
identical to the control, but of course they often can't be coaxed 

I am still relatively new to this business so I could use all of the
advice I can get. 

Here are some potential causes that I think could be contributing to this
problem...what do you all think??

--I am using aliquots of loading buffer that were made up at least 8
months ago. 

--We have recently (a couple of months ago) begun using Long Ranger gels
at 5.75% concentration. They have been working great up until this point.
I didn't create a new matrix file for this since it didn't seem 
necessary. 

--I increased the PMT voltage by 25 volts on the machine around 23 March
because it looked like the blue line on the scan window was too low. Now
it is around 950. 

Other that that everything else seems to be the same. The TBE is made the
same way and very carefully, We are using the ABI Dye-Terminator cycle
sequencing kit, and Centri-Sep columns (often re-used with Sephadex 
G-50). 


Again, any and all comments are appreciated...I still have a lot to learn.


Thanks so much.

Steve

PS sorry if this gets posted twice...I had trouble sending it the first
time.

Steven F. Mullen
Assistant Scientist

Microchemical Facility
University of Minnesota
2-167 Moos Tower
PO Box 206 UMHC
Mpls MN 55455
612-626-3432




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