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Incubation conditions of T4 DNA ligase - (Nov/08/2005 )

Hi does anyone have any suggestion for the incubation conditions of T4 DNA ligase.
I usually do 16-18hours at 16C. And it works but it would be quicker, I would save one day of work if I could do it at higher temperature for shorter incubation time.

The plasmid DNA is 6.5kb and the insert only 0,5kb.
I will cloneusing BamHI and EcoRV sites.



i succesfully get clones in 30° 2 to 8h


Oh, You do?

In the product description of the T4 DNA ligase I use says

Incubate the reaction at:
room temperature for 3 hours, or
4°C overnight, or
15°C for 4–18 hours.

"There is considerable latitude in the temperature and time needed for successful ligations.
The optimal temperature for a ligation is a balance between the optimal temperature
for T4 DNA Ligase enzyme activity (25°C) (1) and the temperature necessary to
ensure annealing of the fragment ends, which can vary with the length and base composition
of the overhangs." (in Promega)

Is not writen that 30C incubation is an option.
But I might next time try my usual incubation 16hours 16C
and also try for the first time , 22C for 3 hours.

Thanks for replying

QUOTE (fred_33 @ Nov 8 2005, 10:40 AM)
i succesfully get clones in 30° 2 to 8h



I haven´t tried 30C, I always thought that the optimum temperature was around 25C for T4 ligase so have always kept it below that. I find in favourable ligations ie. cohesive ends small insert I can ligate for as little as 30min-1hour at room temperature. For blunts or problematic ligations I always do overnight incubations.




my boss got cloes by ligation at 37°. He just start in the morning and finish around 17h30pm. Following: precip and electroporation...

i do normally as him but as i care to temperature i did not go more than 30°.
But on the bench works as well.