Punjab Agricultural University
Question
Asked 21 September 2016
Hi all, for a ligation reaction, is it true that incubating the reactions overnight at 4 deg Celsius would yield the maximum number of transformants?
Read this in a protocol for pGEM-T Easy Vector Systems.
Normally, we would incubate a ligation reaction for an hour at room temperature, so I am curious if there is an explanation behind incubating it overnight at 4 deg Celsius. Have anyone tried it before?
Most recent answer
As explained by Mr Liang, incubating ligation mix at 4 C overnight helps to keep cohesive ends anneled while enzyme works although slowly to covalently seal the cohesive ends. However this may not be true in case of blunt end ligation. However the presence of PEG at lower temperature may help to better hold the blunt ends. Inthe last it the practical feasibility of handling many steps in a day and overnight incubation helps to provide a more libral time to manage many experimental parts.
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Popular answers (1)
Indian Institute of Science Bangalore
Dear Liang,
Any physiological enzyme can be expected to work at 37 degree Celsius as most living organisms physiological temperature is 37. But when we try to conduct these enzyme catalysed reaction (like restriction digestion, unwinding by helicases etc.) in vitro we need to mimic the physiological conditions.
In case of ligation, although most enzymes would follow the bell shaped curve and exhibit optimum enzymatic proficiency at physiological temperature, in vitro if we incubate at this temperature it would increase the kinetic energy of the reaction mix and that would increase the random motion of DNA ends (which needs to come together for joining). This won't allow the cohesive ends to come together for forming the H-bonds. Inside the cell there are other factors which assists and hold/bring the DNA ends together and hence the enzyme is capable of resealing them even at the higher temperature. So, when we set up the ligation reaction we prefer lower temperature which will invoke very less random motion in the reaction mix, allowing the cohesive ends to find their cognate partners and remain as such by the time ligase does its job. As, the enzyme activity would be sub-optimal at such lower temperature, increased incubation time is provided to achieve maximal ligations and balancing between compromised enzyme function and reaction feasibility. So, if you want to use 37 degree you might just incubate for 5-10 mins. But here, enzyme would reseal anything that comes into its way randomizing the outcomes (for eg. insert concatamers can be formed). Thus, ligation is carried out at varied temperatures like 4, 16, 22, 25, 37 degrees and for different time like 24 hrs, 16 hrs/overnight, 4-6 hrs, 2 hrs, 10 mins respectively. All are standardized to suffice specific requirements, you can choose according to yours. You can read the following.
Hope it helps!
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All Answers (8)
Max Planck Institute of Biochemistry
The temperature for ligation mostly varies between 4-16 degree celsius depending on the manufacturing company. NEB suggest us to use 16 degree celsius o/n, Promega says 4 degree o/n. And it is true that Incubating at 16 degree celsius o/n increases the number of transformants in my case when I used NEB T4 DNA ligase. Since the enzymatic reactions are temperature dependent, only under the optimal temperature catalytic activity of the enzyme is high. So for T4 DNA ligase the catalytic activity at room temperature might be less than when compared to 16 degree celsius. The time also has a role to play as if you don't incubate longer you are in half way of the reaction which result in only few transformants.
Hope this helps you
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ligation temperature depends on the nature of the DNA ends you ligate. cohesive ends need annealing before actual ligation and thus incubation at low temperatures is necessary. blunt end ligations can be done at room temperature whereas cohesive end ligations are usually done at 12-16 deg C
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German Cancer Research Center
Ligation is temperature dependent process. It also dependent on ends of DNA used and length of DNA.
Blunt ended DNA ligated better at 4 degree Celsius than 16 degree Celsius.
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Indian Institute of Science Bangalore
Dear Liang,
Any physiological enzyme can be expected to work at 37 degree Celsius as most living organisms physiological temperature is 37. But when we try to conduct these enzyme catalysed reaction (like restriction digestion, unwinding by helicases etc.) in vitro we need to mimic the physiological conditions.
In case of ligation, although most enzymes would follow the bell shaped curve and exhibit optimum enzymatic proficiency at physiological temperature, in vitro if we incubate at this temperature it would increase the kinetic energy of the reaction mix and that would increase the random motion of DNA ends (which needs to come together for joining). This won't allow the cohesive ends to come together for forming the H-bonds. Inside the cell there are other factors which assists and hold/bring the DNA ends together and hence the enzyme is capable of resealing them even at the higher temperature. So, when we set up the ligation reaction we prefer lower temperature which will invoke very less random motion in the reaction mix, allowing the cohesive ends to find their cognate partners and remain as such by the time ligase does its job. As, the enzyme activity would be sub-optimal at such lower temperature, increased incubation time is provided to achieve maximal ligations and balancing between compromised enzyme function and reaction feasibility. So, if you want to use 37 degree you might just incubate for 5-10 mins. But here, enzyme would reseal anything that comes into its way randomizing the outcomes (for eg. insert concatamers can be formed). Thus, ligation is carried out at varied temperatures like 4, 16, 22, 25, 37 degrees and for different time like 24 hrs, 16 hrs/overnight, 4-6 hrs, 2 hrs, 10 mins respectively. All are standardized to suffice specific requirements, you can choose according to yours. You can read the following.
Hope it helps!
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K&H Personalised Medicine Clinic
Agreeing with all the above said points, Ligase/an enzyme activity will be longer at lower temperatures (4 degrees) than at higher temperature. Coming straight to your question, prolonged incubation (Overnight) at 4 deg will increase ligation efficiency for sure. Especially for manual ligations, and when the insert size is large, I prefer incubation of ligation mix overnight at 4 deg. It always worked and gave more transformants.
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Punjab Agricultural University
The explanations by Ms Tias Saha and Kalyani Palasamudram are more convincing if you have time for O/N incubation. I prefer combing DNA fragments incubate at 65 degree C for 2 min. allow them slowly cool to RT (20-25 degree C) add to ligase + buffer mixture and incubate at 4-10 degree C overnight.
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Tianjin University of Science and Technology
Add your insert DNA to plasmid and PEG400 and keep the tunes at 65 C for 5 min, allow tubes to cool at room temperature and add T4 ligase and its buffer and put the mixture at 16C for 3-4 hours and then at 4 C overnight, iit is followed for blunt ends especially.
Punjab Agricultural University
As explained by Mr Liang, incubating ligation mix at 4 C overnight helps to keep cohesive ends anneled while enzyme works although slowly to covalently seal the cohesive ends. However this may not be true in case of blunt end ligation. However the presence of PEG at lower temperature may help to better hold the blunt ends. Inthe last it the practical feasibility of handling many steps in a day and overnight incubation helps to provide a more libral time to manage many experimental parts.
1 Recommendation
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