Help-DIG in situ

Dr. Kent Nastiuk nastiuk at rockvax.rockefeller.edu
Thu Jun 22 14:34:48 EST 1995


pwhitela at uoguelph.ca (Philippa Whitelaw) wrote:
>I am having problems with doing in situs with Dig. I used to have it off 
>pat with 35S but need to use Dig because of the tissue and cell type I am 
>looking at now.  My template is OK and a northern worked ok with the 
>radioactive riboprobe. I am not sure of the Dig labeling of the 
>riboprobe however and am almost convinced that this is where my problem 
>lies.  I am using reagents from Promega and then BM dig- is this a 
>mistake or should I use the premixed stuff from BM?   The most galling 
>thing about all this is that it has worked twice at the begining when i 
>led a charmed life.  I have played with almost every variable in the 
>actual in situ protocol with the same results- absolutely nothing.  What 
> [riboprobe] are people using- I have tried 200-500ng/ml.  Anyway I am 
>tearing my hair out and would be very grateful for advice before I leave 
>science for good! I bet it is something really simple.  Thanks. 
>
>Philippa


since one good synthesis reaction of a DIG labelled riboprobe is 
sufficient for many, many in situs, i routinely do two controls.

1.  spot the sephadex-cleaned probe onto nylon at increasing dilutions   
     and develop the filter to insure good incorporation of the label
2.  spot the template DNA onto a filter at increasing dilutions and      
hybridize with your newly sythesized probe.

you should be able to see sub-pg quantities of your probe directly, and 
in the same range for the hybridization.

if these controls do not work, then the problem lies in the synthesis 
reaction, if yes, the in situ protocol.

good luck





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