Differential Display of Artefacts?

SHO1 sho1 at aol.com
Tue Dec 27 08:50:15 EST 1994


What does it look like when it's not working?

Well, let's start with what it looks like when I think it's working. A
positive pair of lanes seems to be those which contain a set of bands with
identical migration and intensity, as well as some bands of different
intensity. I take comfort in those bands that appear identical, and think
of them as internal controls for similarly-abundant transcripts.

Then there are pairs of lanes where one member of the pair has almost no
distinct bands, or perhaps one or two, while the other has many. Or those
where there are only a handful of distinct bands in both lanes. This
effect seems to be primer-dependent, and I wonder if it represents biology
or rotten primers. For example, on a single gel we'll see a pair or two of
the "good lanes," and several of the lousy ones. Furthermore, reactions
from a particular prep of RNA (we're only using two preps, call them + or
- ) yields both good and poor-looking lanes, depending on the primer pair.

We have also observed that day-old RT products don't amplify the same as
fresh cDNA. Has anyone else found this kind of instability?



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