more ligation problems! - (Jul/14/2006 )

Hi! I´m having problems with blunt end ligation too and i´m getting a little bit desperate by now...I´don´t know what else to try so I hope some of you can help me. The thing goes like this:
I need to clone a Tet resistance gene inside a haemolysin gene which I have already cloned into a vector (this I did by using the TOPO kit). I got my vector by PCR with Pfx from Promega by using internal primers to the haemolysin. This means that, supposedly, my vector has no overhangs (completely blunt ended) and no 5´phosphates. I purified this vector from the gel by using a gene clean kit. I got the tet R gene by digesting a plasmid with Sma I and purified it by gene clean as well. Later I mixed the insert and the vector in a 8:1 ratio, transformed E coli TOP 10 competent cells, plated them onto a LB+tet agar and nothing happened...not even a single colony. I did this like a million times trying to change the DNAs, repurify6ing things, reconcentrating them and still nothing...what is it that I´m doing wrong?

Thanks in advance
Salomé

r u sure that your E.coli expressed the tet resistance gene you inserted? or have i misunderstood the thing?

OK, from what you said, you have cloned the TetR gene inside the haemolysin gene, right?

Did the plasmid transfect OK? Does it have a different selection gene, say, amp? Test this by plating onto Amp plates (simple, I know, but sometimes troubleshooting is like that...), and growing up any colonies, then PCRing for Tet or haemolysin.

Is the haemolysin construct expressing? Test this by in vitro trancription/translation.

Is the Tet gene in the right frame? If the answer to the above two questions is "Yes", I'm guessing the answer to this one is "No", but I have to ask.

Not yet! I have my haemolysin gene cloned into a plasmid, and now i am trying to clone the tetR gene inside this cloned gene. The vector has and amp and kan genes (already tested, besides i have the map-it´s a comercial vector)

I have performed transfection controls with the plasmid in which i have the haemolysin (which i have already quantified) and my competent cells are working properly (many colonies grow in LB-amp plates)...this is why that i am afraid that if i grow the cells transfected with the ligation mixture on lb amp plates i will have to test thousands of colonies to, at the end, find out that they are all background...

I really don´t know if my haemolysin gene is expressing, but since i don´t need expresion of the haemolysisn and the TetR gene I have has it´s own promoter and termination signal, theoretically there should be no problem...right?