TOPO CLONING - (Aug/09/2006 )

I need to insert two different genomic fragments, of 3 kb each, into TOPO blunt vector (separately). The two inserts are amplified by PCR; the first one is easily subcloned into topo, while i've been trying to obtain some positive clones with the second insert for 6 months and still get none !
I have been thinking that maybe there is some secondary structure in my second fragment which causes problems when trying to ligate into the plasmid....
So I've been trying different things :
-adding DMSO to the fragment before ligation
-heating (50o)the fragment before ligation
-ligating for longer periods than recommended by the manufacturer

Eventually, I want to cut it out of TOPO and insert it into another plasmid, so I am beginning to think that even at this step I am going to get all those troubles again....

Can someone give me some advice about this ? Any idea would be very appreciated...

I’m not sure, but I don’t think second structure is your problem… I’ll do the assay using a positive control (may be the fragment you cloned first). And definitively I’ll use a new kit….
Good luck!!

Thank you for your interest, but when I said its working for the first fragment and not for the second, I meant AT THE SAME TIME (and with the same kit) !!
In fact, I tried the two in parallel and the first one I get lots of positive colonies (around 50), while for the second I get 2-3 negative colonies !!
Any more ideas before I get crazy ?

Ok so no one answer to this, I have another question related to this subcloning. Is it possible that my real problem is not really in the ligation of the amplicon in the plasmid, and thus my competent cells could be source of this kind of problem ? I mean, I read about competent cells and I saw that some competent cells specialized for cloning of unstable DNA exist...Do you think this might help ? Has anyone ever experienced this type of problem ? Thanx