Question: Ligation Problem.... - (Oct/20/2010 )

Hi Guys...

I am trying to ligate a PCR product with 2 different restriction sites (Nde I and BamH 1) into a pET 14b vector. The insert corresponds to 1047 bp, while the vector is approximately 5 kb. After my ligation reaction (I use Takara Ligation Mix, which includes everything is needed, Buffer, Enzyme, ATP, e.t.c.) I do my transformation and I streak plates containing the appropriate antibiotic. I also use 2 controls, 1 with the linearized vector and 1 with the linearized vector and the ligase without insert. The next day, I see no colonies to the control plates and I see about 12-15 colonies to the sample plate (this which was streaked with the ligation reaction: insert-vector-ligase). After MiniPrep and the isolation of my vector, I digested the vector with the 2 restriction enzymes, in order to determine and verify the ligation of my insert. The outcome is that I see my vector linearized and I see definitely a shift on the gel, from 5 kb up to 6 kb, but I do not see my insert. In other words, I think that my insert is successfully ligated but some reasons, afterwards only the 1 of 2 enzymes can digest the restriction site. Maybe there is a modification to 1 of 2 restriction sites after the ligation, resulting to this bizarre outcome. And I am sure that the insert has been ligated because of the difference between some of the colonies whose plasmid after digestion run at 6kb, and them whose vector after digestion run at 5kb. It is quite clear on the gel. I was wondering, what might be the reason for this phenomenon...Under these circumstances, one cannot have an efficient vector for expression. Does anybody has any suggestions about this problem? It would be very helpful to me. By the way, the previous days I successfully ligated another gene into the same vector with lower concentrations of vector and insert. Do you think this might play a key role to the whole procedure? Thank you very much in advance for your attention and I apologize for the long text.....

So, your colonies have plasmids that are linearized by each enzyme (separately) producing a 6Kb band, and with both enzymes you see a 5Kb band, but no 1Kb band. Is that right?

If so, then likely you can't see the band for some reason (too little DNA, possible migration of EtBr opposite direction from the DNA, leaving the gel unstained in the low mass range).

Thank you very much phage 434 for your reply....
But actually, the first time (yesterday, when I posted my question), I only digested my vector with both enzymes and I got 6kb band. Today, I thought that it would be much contributive to test each enzyme separately just for control and simultaneously to repeat my double digestion. Wo knows! But in any case, thank you very much for your reply phage 434...