Problem with 3 way ligation - (Mar/27/2013 )

I read through the thread that was pinned on this forum, and a 1:1:1 ratio is something I will try, but I just wanted to post my particular circumstance in case I'm missing something else.

I have a 5 kb destination vector cleaved with EcoR1 and Xho1

A 700 bp gene, cleaved from a TOPO TA vector with EcoR1 and Xba1

and

A 1000 bp 3' UTR, cleaved from a TOPO TA vector with Xba1 and Xho1.

I treated the digested destination vector with CIP to prevent self-ligation.

After a 3 hour, room temperature, ligation with NEB T4 ligase, I transformed and plated. ~100 colonies were attained from 5% of the transformation.

I'm certain my restriction enzymes cut my products only once (ran controls); however, when I try to cleave the plasmids, acquired from the colonies, with EcoR1 and Xho1 (theoretically, cutting out the gene and 3'UTR), I get a single band with a lower molecular weight than the destination vector (~3500).

I just don't see how anything could recombine to create an antibiotic resistant construct that is lower than the actual destination vector.

You should not need CIP, and I would avoid using it. If you want to reduce your vector background, then you can amplify the vector with PCR using primers that contain a restriction site of your choice, then cut with DpnI to cut the template. If you use small amounts of template, then background is very low.

Thanks, Phage. I know, I shouldn't need CIP because the two sticky ends aren't complementary; however, in every iteration of this procedure I tried the destination vector seems to religate. Something isn't right...

I will look into that Dpn1 method, though.