Kpn I site blunting

brett brett at BORCIM.WUSTL.EDU
Tue Nov 21 11:35:42 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

I have seen KpnI (or a contaminant thereof) nibble at the ends, but rarely.
Overall, its performed well. However, I suggest doing very controlled rxns,
ie no need to overdigest. Another thing, Klenow does contain 3'->5' exo
activity. Try T4 DNAP, and make sure you have plenty of free dNTPs for all
fill-ins. Good luck.


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




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