Ligation failed? - (Nov/19/2006 )

I isolated one full-length gene. I put the XbaI site for further expression construct. The expression vector is modified pBINPlus with CamV 35S promoter. Originally, I digested the vector and Plasmid DNA with the full-length gene with enough XbaI for 12 hr, I gel-purified the full-length gene since the TOPO vector which the gene linked with has the same resistant marker (Kan+) as the pBinplus expression vector. I also dephosphorated the digested-pBinplus vector. After ligation, the control (only the dephosphorated vector without ligase) gave lots of colonies, indicating the digestion of pBinplus vector is not completed. I repeated this and the result was same. Later on, I gel-purified the digested pBinplus vector and then dephosphorated it. After transformation, there are not colonies for control and samples at all. Any suggestion is welcome.

Hi beaver72,
Could you give me a detail information on your digestion and ligation reaction? I think you have a problem of or digestion or ligation step. In additon, you should take care of using phosphatase to dephosphorate your vector. I often incubate at 37oC only in 01 hour with phosphatase.
Nam

Xba I is one of the restriction enzymes blocked by Dam methylation. Dam methylase is a bacterial enzyme that methylates the A residue in sequence GATC. If a methylated A is present within your recognition sequence (TCTAGA), it will block the restriction enzyme and stop it working. What you have to do is check whether the recognition site for Dam methylase (GATC) is overlapping the Xba I sites in your vector and your insert. The first two bases of the recognition sequence are present in the 5th and 6th bases (GA) of the Xba I site, but you have to check whether the last two bases (TC) are present in the specific sequences within your vector and insert. If the GATC site is present, the A will be methylated and Xba I will not work. The other major methylase is Dcm methylase, which recognises CCWGG. Strains are readily available that contain knockouts of these genes so that methylation-sensitive restriction enzymes can be used. Hope that helps. Rob