2 replies to this topic

[arwen76]

Hi,
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.

Arwen76

[jadefalcon]

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

formular:

((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

mike

[george@CASE]

the ratio is molar ratio, not volume.

follow the formula given by jade falcon.