for some time now I have been doing radioactive ISH using 34nt long mRNA probes. I label my probes by incorporating 35S-A on an A-tail..
Now my question.. When I design my probes, I normally isolate the mRNA segment that fits my criteria and the reverse complement of this is my probe.
But recently, I made some probes using the complement instead (don't ask why, point is I thought it wouldn't make a difference..). These didn't seem to work well.
So now I am wondering: Does the orientation of your probe matter (alas complement or reverse complement)? I thought that as long as the probe is complementary the bases will still interact with the mRNA and bind! And it shouldn't influence the A-tailing either should it? (it will just flip sides)
Keep in mind I am not talking about in vitro transcription or anything, these probes are chemically synthesized!
Edited by biobio, 07 January 2010 - 04:41 AM.