ligation ratio insert-vector - (Oct/02/2006 )
I am trying to ligate my gene 462bp to a PETM11 (6kbp)vector, I'm using the T4 DNA ligase of Invitrogen and I have some questions about the ligation process and I don't know if somebody could help me.
I want to know if it is enough time 2 hours at 24ºC for the ligation reaction wirh cohesive ends.
Besides, when do you calculate the ratio between insert and vector, what is the best way, using the ratio in molar or in grame proportion. And which is the best ratio when the vector and the insert have different size.
Finally, which is the most appropiate amount of ligation reaction to transform competent cells.
Thanks for your collaboration!!!!!
Sticky end ligation is remarkably fast. It is mostly over in about 10 minutes at RT. Unless you feel like going to lunch, I don't think you need 2 hours. The correct ratio is 1:1 molar ratio (See Sambrook e.g.). It cannot be otherwise, since the ligase does not know which fragment is a vector and which is an insert. If you have inefficient cutting of the vector or insert, these inefficiencies can change the ratio that may be optimal, but if your cutting is good, 1:1 is the answer. We find that > 5% ligation mix in the chemical transformation is inhibitory, so use a maximum of 2 ul with 50 ul of cells (we normally scale this to 0.5 ul in 15 ul of cells).
I agree, you definitely do not need two hours using that ligase. However, for the ratio, I have used a variety of combinations with equal success. I usually run a small amount of my vector and insert on an agarose gel and visually compare intensities. If one is noticeably fainter, I will increase the amount of it that I use in my ligation. Additionally, keep in mind that your insert is much smaller than your vector. Therefore, when you are looking at your gel, equal intensities is actually reflective of more insert fragments than vector. I like to use more insert than vector in my ligations, but this usually means the same actual volume of each (due to what I described).
We ligate for 30 min at RT. It is more than sufficient. We have also tried 15min. ligation and it worked. This is for sticky ends. If blunt u need to increase ligation time
for ratios, I usually use 1:1 or 1:3 . Sometimes even 1:10. it works.
Thanks everybody for your advice, I will take all of them in consideration and I hope that finally I will obtain some colonies!!!
If I have more quesion, I will come back again.
Hi everybody again!!!
Finally I obtain some colonies, at the end the solution was the E. Coli strain, if I changed it, I obtained some colonies, only three, but....
The problem now is other....when I try to growth these colonies in liquid medium with antibiotic they don't do it!!!!
Anyone can help me, please!!!! Because I will back crazy.... perhaps is a problem that my product is toxic for the bacteria...what do you think?
well. probably your plates don't contain enough antibiotics (in comparison to your LB medium in which you try to grow the colonies)... as to a good ligation: I really like the protocol from Invitrogen.