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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