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E. coli ligase is not the way
Posted by perneseblue,
01 September 2010
·
99,369 views
multiway ligation
Dear Blog,
It has been a case of "I did warn you."
I have attempted to improve the efficiency of my multiway ligation by using a different ligase, a higher fidelity ligase. The only other ligase sold commercially is E.coli ligase. It is stated that E. coli does have higher fidelity compared to T4 ligase, however NEB did warn that this ligase was not to be used in cloning.
And yes, I did go and find some E.coli ligase and yes, I did try it out on a cloning. Sadly, E. coli ligase isn't all that good. A standard 5-way ligation failed to produce any useful colonies using E. coli ligase.
Alas, I ended up doing double the work (Identical ligation using T4 and E.coli ligase were done in parallel) to obtain my clone.
So, E.coli ligase is not the way forward.
It has been a case of "I did warn you."
I have attempted to improve the efficiency of my multiway ligation by using a different ligase, a higher fidelity ligase. The only other ligase sold commercially is E.coli ligase. It is stated that E. coli does have higher fidelity compared to T4 ligase, however NEB did warn that this ligase was not to be used in cloning.
And yes, I did go and find some E.coli ligase and yes, I did try it out on a cloning. Sadly, E. coli ligase isn't all that good. A standard 5-way ligation failed to produce any useful colonies using E. coli ligase.
Alas, I ended up doing double the work (Identical ligation using T4 and E.coli ligase were done in parallel) to obtain my clone.
So, E.coli ligase is not the way forward.
