EST clones as probes

Thomas Newman 22313tcn at ibm.cl.msu.edu
Mon Jul 29 17:40:19 EST 1996


Dear Marc,

  The EST is only a partial sequencing of the cDNA in the clones.  For the
PRL2 library, more than 60% of the ESTs are essentially full length.  The
published data is usually on the first 400 base of the 5' end.  I do not
understand why these inserts should not work.  You do not say how you make
your probes, but I have not heard of other folks having trouble with these.
 

Sincerely,
Tom Newman
Michigan State University


In article
<Pine.HPP.3.95.960729100600.25257A-100000 at raven.csrv.uidaho.edu>,
corte922 at UIDAHO.EDU (Marc Cortese) wrote:

> 
> Hello fellow Arabnetters,
> 
> 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.  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.  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. 
> 
> Is anyone using EST clones as probes for northerns?  If so what are the
> disadvantages of using them? 
> 
> Thanks much,
> Marc
> 
> =================================================================
> Marc Cortese                                  Lab: (208) 885-9431
> Department of MMBB                           Home: (208) 882-8810
> University of Idaho                           Fax: (208) 885-6518        
> Moscow, ID  83843                     e-mail: corte922 at uidaho.edu
> =================================================================
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