DIG labelling and detection

[Mary Clare Tracey]

ELIZABETH GALE wrote:
> 
> Hi, I've had some problems with DIG labelling using random priming where I
> get a low yield of probe. I switched to a PCR labelling mix from Boeringer
> and it works really well (200ng/ul probe). You might want to give it a
> try.

I've used the Boehringer 3' DIG-labelling kit and have about 60-70% 
recovery. The probes are never very 'hot' though and I always end up 
having to use far more than the various methods suggest (at least 
twice or more the number of pmols) to get a readable autoradiograph 
result. This increases the cost and the irritation. How does this PCR 
labelling mix work? Do you have to design your own primers to create a 
probe? How small can the probe be (mine are usually around 20mers)?

Regards

MC 
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Mary Clare Tracey		email: maryclareatkokako.wnmeds.ac.nz
Wellington     **NOTE: change the at to @ for reply. Avoiding spam!** 
New Zealand