Ligation of insert to itself problem (View forum version)


Posted 30 January 2012 - 11:14 AM

Im having issues ligating my insert into pet-28a. I get fine digestion using NdeI and BamHI but when i go to ligate it seems as though my insert is being ligated to itself due to the appearance of multiple bands in my gel, seemingly being multiples of the insert. What should i do to get it to ligate to my vector instead of itself?


Posted 30 January 2012 - 01:48 PM

You don't really care about the DNA that ligates to itself. You care about the (possibly rare) event of it ligating to a vector. This is selected for when you transform. Most people don't bother to look at gels of their ligations, and they don't much matter for cloning, except as an analytical tool.


Posted 01 February 2012 - 07:23 AM

If you're that worried about it you can try using an alkaline phosphotase on the insert before you ligate it to the vector, this removes the free phosphate on the 5' end of the insert meaning it cannot ligate to itself, but only to a piece of DNA which still has it's free phosphate, i.e. the untreated cut vector.

but as phage said, if you are using 2 different RE it shouldn't matter as ligation to itself produces unviable bits of DNA which even if they get transformed into a cell cannot do anything.


Posted 01 February 2012 - 09:02 AM

How many colonies in your ligation plate compared to control? This is the question one should ask.