Kpn I site blunting

David de Graaf BMGraaf at dapsas1.weizmann.ac.il
Tue Nov 21 08:20:10 EST 1995


In article <30B01433.5073 at mfour.med.kyoto-u.ac.jp> Hirata,
hiratamz at MFOUR.MED.KYOTO-U.AC.JP writes:
>Hello!
> I have tried to blunt a Kpn l site by Klenow fragment and ligate a 
>PCR fragment into the blunted site.
> When I sequenced the ligation part, the site contained 
>	 GGxxxx (xxxx being PCR primer sequence) instead of Gxxxx.
> Since Kpn I site is GGTAC|C, 
>I expected it to become single G at its 3' end after blunting with  
>Kpn I.
> However, that is not the case. 
> I have sequenced seven independent clones and got the same result.
> Does this mean Kpn I site is actually GGTA|CC?
> This may be a FAQ, but I could not find any bionet document from 

>bionet gopher search engine.
>Thanks in advance for any information.
It is far more likely that during PCR you added an extra base OR that
your primer contained this extra base. PCR tends to add an extra base,
usually an A, but also other bases. I would vote for a mistake in your
primer first, and base addition (since it is not an A) second.

David de Graaf

Department of Membrane Research and Biophysics
Weizmann Institute of Science
Rehovot, 76100
Israel
tel: *972 8 343 686
fax: *972 8 344 112



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