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DNA concentration for ligation - (Jun/13/2005 )

blink.gif Hi!

I read that the maximal DNA concentration for ligation should be 1-10 ng/ul of reaction. Is that true? Somehow I have the impression that the more DNA you have, the better the results are...
Is it reaaly useful to heat insert+vector DNA at 65°C for 5 min before pipetting the ligation? Do you guys do it? I read about it last week , but I had never heard of that before...¨
Bye bye and thanks wink.gif

-giogio-

Hi,
I dont think as less as 10ng/ul is more for a ligation rxn. but even more is not merrier! It all depends on vector ,insert size, type of ligation etc. Heating @65 for 5` is done to break the weak hydrogen bonding between compatible ends.

-Molonco-

biggrin.gif Thanks Molonco!

I have a 14 kb plasmid, and a 2.4 kb insert. I digested both with KpnI, and then dephosphorylate the plasmid. It should not be that difficult, but it did not work.. What total concentration would you use for such a ligation? You think electrocompetent cells would do a better job than chemical competent ones for such a big plamid?

Thanks a lot!

-giogio-

hi
using promega enzyme i use 50ng in 10µ reaction mixture... So it's 5ng/µl.
Using T4DNA ligase, it's 100ng in 20µl... again 5ng/µl...
Before adding enzyme and buffer, i heat sample 65° for 5' and i get better results.
The major point is the ratio vector/insert (molar/molar), and can be up to 1:12.

-fred_33-

Yo, what Fred said... except I start with 50ng of vector, then calculate the mass of insert to add based on the ol promega fomula.

Oh, yeah, after ligation and before transformation, heat the ligation rxn to 70C for 10min, then quench on ice, spin and add to your bugs.

-pBluescript-

QUOTE (Molonco @ Jun 13 2005, 05:02 AM)
Hi,
I dont think as less as 10ng/ul is more for a ligation rxn. but even more is not merrier! It all depends on vector ,insert size, type of ligation etc. Heating @65 for 5` is done to break the weak hydrogen bonding between compatible ends.


molonco, did u mean heat @65 for 5' before the ligation?

-whimsicalDNA-

I found that cutting with KpnI causes a lot of problems with my cloning. I could never get it to work because it never really cut completely. I switched to using Acc65I which cuts the same sequence as KpnI but is much more efficient and had no more problems.

-baylorbean-

Thanks a lot!!! I will try once more with kpnI (I already digested...). Then I will try your way!

Have a nice evening biggrin.gif

-giogio-

I'm treating to make a ligation of two blunt ends... what is the right relation insert:vector?... what is the correct enzyme concentration?...

-donisaid-