In situ with DIG-cRNA

Frank Maiwald maiwald at mpiz-koeln.mpg.de
Fri Apr 3 03:17:58 EST 1998


Timur,

I am doing DIG-Northerns and I had similar problems. For convenience, I
am using PCR-products as probes. The PCR also contains linearly
amplified and labeled vector sequences (and the primer annealing sites
on each product). These sequences lead to background hybridization if
the PCR-product is not purified on a gel. If you use primers that anneal
in the vector even these small pieces can lead to background. I think
the DIG-system is just a bit too sensitive in this respect.

Frank

--------------------------

Timur Yarovinsky wrote:
> 
> I am trying to set up in situ to detect mRNAs with DIG-labeled cRNA probes
> but still have very high background with sense probes. I use protocol
> according to Boehringer Mannheim recommendations, tried various
> hybridization and washing conditions (T from 45 to 65C, several washes
> with upto 40% formamide, 0.2x SSC;  treated with RNases A/T1,
> hybridization buffer contains 1 mg/ml tRNA, 1 mg/ml sheared denatured
> herring sperm DNA). Nevertheless, sense probe gives signal as strong as
> antisense one.
> 
> The cRNA probe is 385 bp long (plus a piece from the vector), checked on a
> gel and concentration estimated by dot-blot. Antisense is 100% homologous
> to somatostatin receptor isoform 2, obviously sense has no homology.
> 
> I would appreciate any recommendations.
> 
> Sincerely,
> 
> Timur Yarovinsky



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