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Question about phosphorylation and ligation - (Dec/07/2006 )

Hi,

I have a plasmid that contains a Spec and an Amp selection. In order to do my experiment I need to get rid of the Amp marker. The plasmid had a single NdeI site close to the Amp marker so I designed primers to insert another NdeI site. I did my PCR and digested my product with NdeI producing to bands. One that was the Amp marker and the other band was the rest of the plasmid. I cut and gel purified only the band that did not have the Amp and I did a ligation. After that I tried my transformation but did not get any transformants. I suspect that maybe my plasmid didn't ligate so my question is: Is having the compatible NdeI sites enough to promote ligation or do I need to do some phosphorylation of the ends.

Thanks for the help.

-sramosmo-

QUOTE (sramosmo @ Dec 7 2006, 12:01 PM)
Hi,

I have a plasmid that contains a Spec and an Amp selection. In order to do my experiment I need to get rid of the Amp marker. The plasmid had a single NdeI site close to the Amp marker so I designed primers to insert another NdeI site. I did my PCR and digested my product with NdeI producing to bands. One that was the Amp marker and the other band was the rest of the plasmid. I cut and gel purified only the band that did not have the Amp and I did a ligation. After that I tried my transformation but did not get any transformants. I suspect that maybe my plasmid didn't ligate so my question is: Is having the compatible NdeI sites enough to promote ligation or do I need to do some phosphorylation of the ends.

Thanks for the help.


While I don't quite understand what you are doing to get rid of the amp gene, for ligations compatible ends should be enough. However, I usually add about 15-30 Units calf intestinal alkaline phosphatase (CIAP) to my digesting vector when there is about 20minutes left in my digestion. Don't add this to your insert. Only your vector. This will dephosphorylate your vector to discourage self ligation, and keeping your insert phosphorylated will similarly promote ligation to the vector. Hope that helps!

-Cheamps-