Ligation problem

[Gunnaporn - Veerachato]

Dear Andrew 

               I used to have the problem of the 2 adjacent restriction sites when I used pET16b. The 
restriction sites are NdeI, XhoI and BamHI, respectively and I wanted to insert my PCR fragment at
 NdeI and BamHI sites. I could not ligate my PCR fragment into the vector. I solved the problem by 
inserting another PCR fragment that has BamHI at both ends to extend to length between NdeI and  BamHI restriction sites in my vector. Then  I cut the vector with NdeI and BamHI. By this method I  could make sure that the vector was actually cut and I successfully ligated my PCR fragment to it. 

I hope this can help you to solve your problem. 

Gunnaporn  Veerachato 
vgunnapo at mailcity.com  


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