4 replies to this topic

[alextam]

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

[phage434]

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

[scolix]

You will know when you try ligating with 50ng.

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

[HOYAJM]

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)

[scolix]

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.