reply to smearing on pulse field gel

Ross Bowles Ph 3629487 bowlesr
Wed Mar 8 18:04:48 EST 1995


In reply to Ms WF Hong regarding smearing of one of her PFGE samples;
We have discovered from our PFGE work that there can be considerable 
differences in the quality of digests between individual isolates, within 
the same species. Sometimes these differences are due to poor quality 
extractions (the DNA is not sufficiently purified in the plug or it has 
been sheared). The smearing of your sample may be evidence of shearing, 
so it may be worthwhile to run an undigested portion of your problem 
sample against an uncut portion of a "good" sample to see if there are 
any differences.
Most digestion problems we have observed in our studien appear to be
closely related to the enzyme used. In our studies of Actinobacillus 
pleuropneumoniae, two enzymes cut all 50 of our samples very efficiently,
another cuts 40 out of 50 and the other about 35 out of 50 samples.
Digestion success rates could be improved in our study by preincubating for 
long periods in restriction buffer alone, before adding fresh buffer and
enzyme, or by multiple preincubations in restriction buffer before adding 
the enzyme. 
The fact that some enzymes digest well tends to indicate that the 
samples are relatively clean, however for persistently difficult samples, 
preparing a second sample may be unavoidable. Some enzymes simply appear 
to be highly variable in their ability to digest, while others are very
reliable, so digestion can have some degree of it hit and miss. So try 
more prolonged and multiple preincubation steps, and hopefully this will
improve your results. 
Otherwise, you're options appear limited. You may have to try preparing 
new plugs of the sample, and hope that your original was just a one off,
low quality extraction. If you wish to have a more detailed discussion, 
don't hesitate to e-mail me.

Ross Bowles 
Animal Research Institute 
Locked Mail Bag No. 4 
Moorooka  Qld  4105
Australia







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