insert to vector ratios for ligations - what do y'all do? (Jun/27/2006 )
OK so i'm new at cloning and have only been doing it for about a month... and never successfully. The first people i worked with taught me to always use more insert than vector while setting up ligations, so i've always made ligation reactions with I:V=3:1, 5:1 or 6:1, trying both just using ratios by mass and actual molar ratios. then today i was working with someone new who has more experience in the area and was taught that i should use more vector than insert. what do you do? are there certain conditions where i should do one over the other? how do you know what to do - is it just by experience? thanks for the help!
I have been told to use and frequently use 1:1 and 1:3 ratio for ligation - insert being more. But I have also tried 1:5 and 1:10 ratios- insert being more- and it has worked.
I guess it depends on experience as well.
I have learnt that one should use mor einsert than vector. In some cases, when for example the insert and the vector are more or less the sme size, one could try and use more vector. I haven't done it so far but some of my colleagues did it and it worked
A 1:1 molar ratio is the right answer, despite what anyone else tells you. See Sambrook, which says the same thing. I think confusion reigns because of the (supposed) average length differences of insert (1 Kb) and vector (3 Kb), which makes people say 1:3, and then people get confused about where this comes from, and whether it is molar or gram ratios. The ligase *does not know* which of the DNA fragments is the vector or the insert. It can't tell. There can be no reason for the reaction to favor one over the other.
I like to use 1:3 - 1:10 molar ratio (with more copies of the insert).... sometimes cloning needs to be optimized and it is hard to predict, but generally 1:3 has worked in my hands.