optimum temperature for ligation - (May/11/2009 )

sagar on May 15 2009, 03:46 AM said:

T C on May 15 2009, 11:25 AM said:

Hey,

I just take Ice cold water and leave the ligations in this water, which is left at RT for 5-12 hrs. Its been working for the last 5 yrs for all kind of ligations.

Best,
TC

That sounds interesting. What is the company from which you use ligase ? I use ligase of fermentas. My vector and insert concentration is 40ng/ul.i use the universal calculation for ligation

insert concentration (ng/ul)=vector conc X( insert length/vector length)X (insert/vetor ratio)

I have tried a lot of times with 16C and less temperature but all in vain. Should I try enzyme from some other company?

Thats exactly what I use.
NEB ligase.

DRN on May 15 2009, 06:50 PM said:

T C on May 15 2009, 10:25 AM said:

Hey,

I just take Ice cold water and leave the ligations in this water, which is left at RT for 5-12 hrs. Its been working for the last 5 yrs for all kind of ligations.

Best,
TC

at low temp good for annealing ( but differ according to the restriction site, cohesive end with like AATT would anneal more readily at low temp) but u compromise the activity of ligase , the activity fo ligase would decrease at low temp.

At high temp it's the opposite.

Hey,

Honestly, I am not sure how it works but if different kinds of ligation (blunt/sticky) and different enzymes require different temperatures, then these conditions are met. However like hanming86 points out, this is at the expense of ligase activity which follows a gaussian curve. But you don't really care as there is enough ligase in the mix.

Yes I do choose the concentration of vector and insert. I don't quantify it but generally take less vector and more insert and use just what is required. If you take more, you never get colonies due to crowding. I know I have a crude way of working as I run 3 ul on teh gel and visually decide how much of vector and insert to take but it works.

Best,
TC

sagar on May 17 2009, 03:31 AM said: