Afl II linearization & ligation - (Aug/05/2006 )
Hi!
I´m using Afl II to linearize my vector and then using ligation to get my linker to the Afl II site. The reaction should be quite easy in theory but the Afl II cut sites seem to ligate very poorly (they don´t even know in the NEB´s internet site what the problem is). The vector doesn´t even give any background after transforming to DH5alpha. So the question is that has someone used the enzyme in the same way and found out some good tips how to improve the ligation efficiency? I´m 100 % sure that the reason why the ligation isn´t working is in the enzyme. Any ideas?
please clarify for me...have you cut the vector with afl ii, purified, and re-ligated it back to itself as a control?
and, can you be certain that there's nothing in your purification protocol that would inhibit future ligation?
and, are you certain that the ATP in your ligase buffer is good (i.e., the buffer is fresh, hasn't been frozen/thawed too many times)?
and, can you be certain that there's nothing in your purification protocol that would inhibit future ligation?
and, are you certain that the ATP in your ligase buffer is good (i.e., the buffer is fresh, hasn't been frozen/thawed too many times)?
Sorry, if i didn´t make my point clear. Yes, i´ve cut the vector with Afl II, purified it and done a ligation reaction with the linker and at the same time another reaction with the vector it self as a control background reaction.
The purification protocol i use has been the same as with previous ligations with other vectors and inserts and worked out fine so nothing has changed in that step.
Since i´ve used the same ligation buffers and enzymes as before and they have worked out fine and i can´t blame them for not working correctly either.
As i see it the problem is with the enzyme (as they tell it also within NEB`s internet page) that the enzyme cuts the DNA so that there forms somekind of secondary structures that are not so easily ligated even back to each other. Normally i would get some background but not with this enzyme.
So, i was asking in anyone knew any good tricks to improve the ligation reaction with this particular enzyme.
I would try new ligase buffer first
trust me
the ATP is a necessary cofactor and it can go bad