Ligation in PTYB12 vectors ofthe IMPACT protein purification system... - (Aug/31/2001 )
I cannot get ligation and transformants using the PTYB vectors of the IMPACT system, I tried different protocols for the ligation and still don't get any transformant, my competent cells are fine (always run a positive control for transformation using another plasmid and got a lot of transformants). The gene i am trying to clone is the glutamine binding protein of Rickettsia prowazeki, i am thinking it may be due to toxicity of the gene?, or for some reason my ligation is not working at all, I cut both insert and vector with SmaI and NdeI, so i generated 1 blunt end and 1 staggered end, so supposely my vector cannot recircularize, tried vector-insert ratios of 3-1,1-3,1-1, 10 and 20 final volumes of ligation. I tried different concentrations of DNA, and I finally got few transformants using 60fmol(287ng) of the vector and 180fmol(84.97ng) of my insert(726bp) in 20Ul ..... but still aren't this concentrations of DNA too high?, I am using T4dna ligase and buffer from NEB, I would really appreciate is somebody will give a hint of what is going on.... I am desperate...
Try cutting your vector with NdeI and SmaI, then cut the insert with NdeI. Ligate cohesive NdeI sites of insert and vector for 2hrs at room temp, visualise on gel and gel extract the vector-insert fusion (making sure of correct size). Purify this fusion, then ligate overnight at 16C. If you dont get any transformants try electroporation and or increase the DNA concentration.
All the best Paul