EST clones as probes
frist at cc.umanitoba.ca
Wed Jul 31 13:23:31 EST 1996
I am interested in your problem, but I'd like to hear
more details of some of your points:
Marc Cortese wrote:
> I have been experimenting with using EST clones as probes for northern
> expression experiments. I've not been satisfied for 2 reasons. First,
> the size of the inserts have been larger (by about 2X) than the published
> sequence for the clone in Genbank.
Why is this a problem? By definition, and EST is a 1-pass sequence,
so it doesn't tell you anything about the length of the insert.
Shouldn't a longer probe allow you to use a higher stringency?
Second, probes made from the purified
> inserts will not survive a stringent wash (0.2X SSPE, 68 degrees C). I
> get great results using the same blotting, crosslinking, prehyb, hyb,
> labeling, and washing procedures with legitimate full-length cDNA clones
> but no results with any of the EST clones I have tried so far.
An EST is a sequence derived from a cDNA. Why is such as cDNA
less legitimate? It sounds like you're making a distinction
between two different kinds of cloning methods. What is
> I *can*
> get signals with ESTs if I stop washing with 2X SSPE but that is not
> adequate for gene that are members of families.
Do you mean that what you are trying to do is to distinguish
between different members of a multigene family by hybridization
with different short (and presumably member-specific) probes?
> Is anyone using EST clones as probes for northerns? If so what are the
> disadvantages of using them?
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