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Help on ligation of big fragment(9kb). - ideas on good ligases? (Aug/20/2005 )

Hello guys, I am doing a horrible cloning right now, since the insert is 9KB! In the last two months, I got nothing. Ligation was not working at all. I guess the ends cannot meet together. Any ideas on good ligases for such difficult cloning? New england biolab literally provides only T4, if I am not mistaken. Thanks A LOT!!!


I'd recommend that you try a very dilute ligation. Since the long strands are required to ligate to their own end, the probability of this happening goes down dramatically as the concentation of ends goes up. Ironically, the normal reaction is to try to increase DNA concentration when you are having trouble, while for long fragments, low concentrations favor higher yields. Is this a sticky or blunt ligation? Same or different sticky sites? Dephosphorylated or not?


It is a sticky ligation, with same sites on both ligand and vector (pcDNA3.1)...
I am trying to cip it this time. I will see the result tomorrow. It's been such a long time...
Thank you for the suggestion. I will try to diluted it out and see what happens. Maybe use less vector? Typically I use 10ng/rex. What do you recommend on that?