Ligation

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poorani
poorani's picture
Ligation

Hi

I tried to Cut pAB 6XHis with Bgl II  to linearize and I did the same with my insert, I tried to the SAP treatment, for that I did ligation of the cut  vector alone and insert alone in parallel to compare how my SAP treatment went. For the first time i dont see any colonies on the self ligated vector or the insert. I could not figure out why? for some I did use more than 400mg in gel extraction column ( Qigen gel purification kit max- 400mg per column)  can some one  please help out

Thanks
A

Jason King
Jason King's picture
As I understand this, you cut

As I understand this, you cut your vector with Bgl II which linearizes it then you cut out an insert from elsewhere. Did you SAP treat both the vector and the insert or just the insert?

You need to SAP treat the vector, not the insert. THe ligation check using SAP treated vector should not give any colonies. Ligating the insert shouldn't give colonies since the insert doesn't contain any plasmid sequences such as the ORI, required for repication in bacteria.