Problem with 3 way ligation
Posted 27 March 2013 - 05:43 PM
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
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.
Posted 27 March 2013 - 07:26 PM
Posted 28 March 2013 - 01:53 AM
I will look into that Dpn1 method, though.