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ligating blunt ends of a linearized plasmid, is kinase necessary?


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#1 kaveh

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Posted 04 March 2013 - 11:52 AM

A quick cloning question:

I want to delete part of a plasmid. I double digest with to REs which result in sticky ends. I gel-purify the desired linearized fragment. I blunt-end with T4 polymerase and the ligate the ends together by T4 ligase.

Before the ligation, do I need to use T4 kinase to add phosphate groups to 5' ends?

thank you

-k

#2 jerryshelly1

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Posted 04 March 2013 - 02:15 PM

No, T4 ligase is sufficient for blunt end ligation. The provided ATP will provide the phosphate linkage.




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