14 replies to this topic

[sagar]

hi all

im having a doubt regarding the optimum temperature for setting ligation of stick ends ,in general the temperature range between 4-16C in suggested but some companies suggest 22C . But this temperature is more than the Tm value of the sticky ends then how can the dna fragments ligate in this temperature?

Can anyone help??

[phage434]

Annealing of sticky ends is an equilibrium process, with the probability of annealing related to the temperature.  Ligation optimally happens at 37C, but annealing is very ineffficient at that temperature.  Annealing is good at 4C, but ligation is very slow.  Somewhere in the middle, both things happen.  The optimal temperature somewhat depends on the overlap bases.  For EcoRI, with an AATT overlap, the optimal is low, perhaps 10C.  For NotI, with overlap GGCC, the optimal is probably at room temperature or above.  Convenience dictates that room temperature is an easy compromise.  Room temperature for 1/2 hour works well for me with normal ligase buffer (not quick ligase) and 20-40 ng of vector and equimolar amounts of insert.  This also works well for three-way ligations.

[Vini]

phage434, on May 11 2009, 12:55 PM, said:

Annealing of sticky ends is an equilibrium process, with the probability of annealing related to the temperature.  Ligation optimally happens at 37C, but annealing is very ineffficient at that temperature.  Annealing is good at 4C, but ligation is very slow.  Somewhere in the middle, both things happen.  The optimal temperature somewhat depends on the overlap bases.  For EcoRI, with an AATT overlap, the optimal is low, perhaps 10C.  For NotI, with overlap GGCC, the optimal is probably at room temperature or above.  Convenience dictates that room temperature is an easy compromise.  Room temperature for 1/2 hour works well for me with normal ligase buffer (not quick ligase) and 20-40 ng of vector and equimolar amounts of insert.  This also works well for three-way ligations.

Hey I am surprised that ur ligations work at RT kept for a couple of hrs!! Is it just for NotI digests or in general???in our lab, we keep the ligations at ~16C n leave it o/n or for 6-7 hrs.

[phage434]

In my experience, cohesive end ligations will work in 1/2 hour at room temperature, or they will not work at all.  The critical factor is the quality and concentration and relative concentration of the input DNA.  Remember you only need a few molecules to be correctly ligated.  Far more important is to eliminate the background, so that the colonies that appear are the ones you want.  We do this right now primarily by switching antibiotic resistance during the cloning -- assuring that the target plasmid has a different resistance than the source DNA fragments.
But there are lots of other approaches.

[microgirl]

Room temperature, overnight works for us!

[sagar]

microgirl, on May 12 2009, 11:11 PM, said:

Room temperature, overnight works for us!

This is for cohesive ligation ? Does it depend on the enzyme sites that are in the hanging position or it is applied to what so ever enzyme the vector and insert are cut with?
mine is ecori and hindiii cut can i use RT?

[phage434]

I do EcoRI ligations at RT routinely, although it would probably be better to do it at a lower temperature.  Convenience and speed are important.

[T C]

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

[hanming86]

yea my method is quite similar to TC's too. It gives a "continuum of optimality". the temp will keep rising to room temp slowly  at diff temp, it will be optimal for the different condition explained by phage343.

[sagar]

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?

[Vini]

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?

try using NEB T4 DNA ligase under same conditions

[T C]

Thats exactly what I use.
NEB ligase.

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

try using NEB T4 DNA ligase under same conditions

[sagar]

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

hi
im still trying but i am very curious to know how does the ligation work following your procedure ?The temperature of the water  shall increase slowly when you keep at room temperature, how does it help in ligation?
Do you give emphasis on the concentration of vector and insert used?

[hanming86]

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.

[T C]

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:

im still trying but i am very curious to know how does the ligation work following your procedure ?The temperature of the water  shall increase slowly when you keep at room temperature, how does it help in ligation?
Do you give emphasis on the concentration of vector and insert used?