Differential Display of Artefacts?

Michael Cooley szcooley at dale.ucdavis.edu
Tue Dec 27 13:09:19 EST 1994


SHO1 (sho1 at aol.com) wrote:
: 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?


That last problem is very odd. I have gone back to cDNA's I made months 
ago and got good amplification. I have heard of ssDNA going bad due to 
impurities in the H2O. I store my cDNA at -80 oC and usually heat it to 
65 oC before pipeting. Are you using silonized tubes and tips?

In regards to differences between amplifications of the same RNA using 
different primers, I saw the same thing and dismissed it as being due to 
the primer sequence. The real considerations is whether you can use the 
same primer on the same RNA and get the same banding pattern. Also I 
never used any anchor primer which contained a T at the end. This was a 
suggestion from others here on campus who could not get reliable bands 
from these anchors.


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