DIG labeling PCR probes II

Frances Hannan Zoology flh at mole.bio.cam.ac.uk
Fri Sep 23 10:14:36 EST 1994

Brett Beitzel <brett_beitzel at msmtp.idde.saci.org> writes:

>Has anyone had any experience doing PCR DIG labeling for FISH probes?  I
>have been trying this but have not had very good yields.  Also, what are
>some methods people use for cleaing up PCR FISH probes after PCR?

I have been using PCR DIG labeling for Drosophila wholemount in situs. The 
template used is a "normal" PCR product that has been gel-purified (using
NA45 paper - see previous posts on this subject).

200ng template in dH2O
2.5ul 10xPCR buffer (Perkin-Elmer)
5ul nucleotide solution (Vial 6 Boehringer-Mannheim)
2ul PCR primer (antisense strand)
0.3ul AmpliTAQ
Make up to final volume of 25ul with dH2O

Cycling conditions 94oC 45s, 55oC 30s, 72oC 60s for 25 cycles

I never purified the ssPCR product just freeze at -20oC to stop reaction
and then use directly in the hybridization mix. I would stay away from
any phenol or chloroform as they may wreck the DIG but EtOH p'ptation
should be fine.  

I check product size on a minigel blotted to nitrocellulose and probed 
with anti-DIG Ab and always get full length probes (~300bp normally). 

Thanks to Manfred Grabner in the Glossmann lab for this protocol.

Frances Hannan
BILMS, Zoology, Downing St, Cambridge, CB2 3EJ, UK
flh at mole.bio.cam.ac.uk
Frances Hannan                                       
BILMS, Zoology, Downing St, Cambridge, CB2 3EJ, UK   
Phone (0223)336663, FAX (0223)461954                 
flh at mole.bio.cam.ac.uk                               

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