I'm new in the fiel of cloning and I am struggling in the ligation thing...
I would like to know what is the best molar ratio for ligating a 302bp insert into a 2021bp vetor. I read that when you have a small insert, you should use a huge amount of it in order to get a good result. In the other hand, some people say, that you should't, because you could form dimers between inserts. So I'm very confused. I applied the basic formula and did some online calculations, but I got different results everytime.
Is there another way to do it? What is the logic behind insert/plasmid lenght, ligation efficiency, etc ...
Also, I have 40 ng of 302bp insert in 20ul (total) and 400ng/ul of the vector. If the final volume of my ligation reaction is 10 ul, should I increase this volume based on my DNA (if I use all the 20ul) or decrease it in order to stay between 10 - 20ul of ligation reaction? The volumes of enzyme, buffer and BSA should be proportional to the final volume?
Usually, for a 10ul mix (T4 DNA ligase Promega):
0,1 - 1ul enzyme (should I use 1ul?)
2 ul Buffer
Thank you very much!