dkim at nmsu.edu
Sat Apr 22 10:12:39 EST 1995
Re: Not getting bands in differential display.
I have used the differential display technique in the past, and found
that I got better results if I used a lot of enzyme. I had as much as 2
units of Taq polymerase per reaction (50 microliter). You can get away
with 1 unit, but 2 is more reliable.
I don't know why this was the case, but it made the reactions more
robust. It might be that there are more active sites of polymerization
in a differential display reaction, and so more enzyme is needed to
support it. Remember that the standard single-product PCR reaction
starts off making very little DNA in the initial reaction cycles and
works its way up, making one band. The differential display (and other
random-primed methods) will start off making many different DNA
molecules, and amplify each of them. You should need a much larger
amount of enzyme to support it.
If there are "responsible opposing points of view", I'd like to hear
them. I don't really know enough to make a solid case for my opinion here.
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