trouble cloning into red vector - (Apr/27/2007 )
I was looking for some info on dephosphorylation and found this forum, and decided to use the opportunity to get some help. I am ligating two inserts cut with one RE (EcoRI) into pIRES2-DsRed-exptress vector, to create two plasmids with my two genes of interest, but I guess because I only use one RE all my clones come out to be just vector alone, without insert. I tried using roche's SAP to desphosphorylate before ligation and it worked on one of the inserts, but I keep having trouble with the other one. Outside of me going at it over and over again till it finally happens, is there something else I can do to facilitate the process and get the insert into the vector?
Is possible for you to use 2 RE which give two cohesive ends? May be you have to dephosphorylate both vector and insert, since both ends are similar. You try to dephoshorylate with CIAP.