digestion problem

[ummorie1 at cc.UManitoba.CA]

Hello everyone:

	I am working on isolating a resistance gene in wheat using AFLP.
Recently, the first step in the protocol - the digestion of genomic DNA -
stopped working.  So I began testing my digestion reactions on agrose
gels.  The orginal protocol has you digest with one 4 base pair cutter
enzyme (Mse I) then added to the same reaction tube the  second enyzme,
6 base pair cutter (Pst I)(with buffer and water).
So I split the two enzyme reaction up into two different reactions but
kept the concentrations the same.  So I tested 4 to 8 individuals with
both  MseI (also known as Tru9 I) at a total volume of 10 ul with 1 ug DNA and 5
Units of enzyme (2 hour digestion), and with Pst I at a total volume of 50
ul with 1 ug DNA and 5 Units of enzyme.  I also ran a non-digested check
DNA for  each. From this I found out that my Pst I enzyme was not
digesting and that my Mse I enzyme was clearly digesting.  All my
non-digested DNA was clearly intacted (at the top of the gel, no smear)
 
So I tried several things:
- I ordered new enzyme and buffer
-increased the incubation time from 2 hours to 24 and 48 hours
-different 6 base pair cutters (EcoR I and Sal I)
-different final volume: decreased it from 50 ul to 10ul
-newly extracted DNA
None of these things worked.

Now, as I understand it 6 base pair cutters are more sensitive to
contamination such as carbohydrates and proteins.  When I tested my DNA on
the spectrometer it was not as clean as it could have been.  When I tried
to clean up or do a new extraction I was unable to get the DNA any
cleaner.  I suspect that I need to find a different protocol for
extracting DNA.  However, from the start of my work with AFLPs I have been
using DNA stock material aliquots from my orginial extractions.  So in
other word I have been using the same DNA throughout my project (aliquoted
out so that it is not going through many freeze/thaw cycles).  So at one
time this very same DNA was being digested by the Pst I enzyme!  I
understand that DNA can degrade over time, and I have not seen that in any
of my undigested sample.  But can the quality change over time
(intuitively you would think no)? I have preformed spectrometer readings
on the individual aliquotes and there is little difference between one
aliquot and another.

If anyone has any suggestions or explanations I would greatly  appreciate
them.  

Thanks a bunch.

Larisa Morier
Masters Student
University of Manitoba
Winnipeg, MB
Canada