Ligation Problem - (Feb/02/2012 )

Hi all,

I am recently having problems in ligating an insert into a vector. My vector is a 8kbp construct and my insert about 1,5 kbp. However after dephosphorylating my vector i have no colonies at all (Using T4 dna ligase). So I switched to the quick ligase kit, and they recommend to use 50ng of the vector and a 1:3 ratio for each insert. But is 50ng enough? As my vector is quite large, I am not sure whether 50ng of vector would be sufficient for the ligation reaction...

Thanks,

Alex

If you ligation is working well, then this is way more than enough.

You will know when you try ligating with 50ng.

the manufacturer has probably tested dif. DNA conc. and is recommending 50ng.

Promega recommends 100-200 ng of vector DNA for their T4 DNA Ligase. The NEB ligase recommends 50 ng. I think anywhere in these ranges should be fine. Also, try different ratios. In my latest clones, it was not the 1:3 that was optimal, but much higher (1:8)

For cloning into some vectors, you might need to use a very high ratio.

With the NEB quick ligase kit, I follow the instructions and it works.