BRL RNA ladder hybridizes to my probe

Mike Poidinger mikep at biosci.uq.oz.au
Mon Aug 21 17:23:44 EST 1995


stallard at badlands.NoDak.edu (Lana C Stallard) wrote:

>I wonder if anyone has had this happen? I am using Gibco BRL RNA weight 
>standards (made from phage lambda DNA spliced to an RNA transcript). I get 
>significant hybridization with a mouse DNA probe (a cDNA probe of 
>angiotensin converting enzyme) to the ladder when i transfer the ladder 
>onto a Northern blot. Why would there be so much sequence similarity between lambda and a mouse gene? or could my probe still contain some sequences of the vector it was produces in (Bluescript 
>plasmid) which is similar to the phage DNA? My boss is convinced i have a 
>very non-specific probe but i think there must be a reason. 
>	Thanks for any comments or ideas you may have. 
>	Lana Stallard

How did you label your probe?  I once made a cDNA probe by exicisng the cDNA
band of interest from a gel (away from the plasmid) using P32 incorporation with
random primers on the exiced band, and still had enough contaminating plasmid in
my target DNA prep to pull AMP from a (supposed) eukaryotic genome library.  The
same material also bound to BRL's KB ladder.

If you are using random priers, my first suggestion is to throw them away and
use specific primers instead.

Mike


----------------------------------------------------------------------------
Dr Mike Poidinger      Now don't be lazy, 
Microbiology, UQ       with the pleasure of sin  (Nitzer Ebb)
Australia              
mikep at biosci.uq.oz.au  
----------------------------------------------------------------------------




More information about the Methods mailing list