Vector- Insert Ratio in Ligation - (Jan/12/2005 )
usually if you do a ligation it is recommended to use a vector- insert ratio 1:3 in general. I think this works great if the insert is smaller or maybe the same size as the vector. But what if the insert is about 3 times bigger than the vector. Is it then useful to try it the other way round: 3 times the vector to insert?
Any ideas or experiences would be really great. I'm having trouble with one special cloning!!!
Thanks to ya'll.
the formular I have here at hand is from the promega portocols and applications guide "the source for discovery": (I abbreviated a bit)
-estimate the concentration of vector and insert by agarosegel-elpho or OD measurement
-test various vector/insert ratios to find the optimal ratio
-in most cases, a 1:1 or a 1:3 ratio work best
((ng vector)x(kb size of insert))/(kb size of vector)) x (molar ratio of (insert/vector)) = (ng insert)
example: 500bp insert to be ligated with 100ng of 3.0kb vector in a 3:1 ratio
((100ng vector)x(0.5 kb of insert))/(3.0 kb vector)) x (3/1)) = (50 ng insert)
hope that answers your question
the ratio is molar ratio, not volume.
follow the formula given by jade falcon.