help:double enzyme digests,ligation,but transformant wrong.w

[jecobsen]

help:double enzyme digests,ligation,but transformant wrong.w

• Quote

Post by jecobsen » Thu Jun 29, 2006 8:19 am

NdeI and SacII double digests and then divided my vector and segment(both 3kb),ligation at 4degree. finally,got several transformants(6kb).select two enzymes(both vector and segment have their sites) cut these transformants,only got 6kb segment, no small segment.I selected other two enzymes to check it, the consequence is the same.

I want to construct other vector at the same time with the strategy, this time I got the small segment,but the orientation is wrong.

why?

thanks

[oppox]

• Quote

Post by oppox » Thu Jun 29, 2006 9:42 am

how small fragment should u get? and do u compare the size of the plasmid with the original 6kb plasmid or du u compare it with a ladder? Why I ask is because sometimes an open vector travells slower (on an agarose gel) then a closed one, so maybe it compensates the smaller size?

[canalon]

• Quote

Post by canalon » Thu Jun 29, 2006 1:51 pm

your only fragment in the 2nd digestion is 6kb, so it looks like you have a single digestion. Have you tried to cut independantly with each Restriction enzyme? Maybe one of them is not working.

[jecobsen]

• Quote

Post by jecobsen » Fri Jun 30, 2006 1:47 am

how small fragment should u get? and do u compare the size of the plasmid with the original 6kb plasmid or du u compare it with a ladder? Why I ask is because sometimes an open vector travells slower (on an agarose gel) then a closed one, so maybe it compensates the smaller size?

about 1kb.compare the size of the plasmid with the original 6kb plasmid.