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Vector- Insert Ratio in Ligation


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#1 arwen76

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Posted 12 January 2005 - 01:00 AM

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

#2 jadefalcon

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Posted 12 January 2005 - 06:33 AM

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
--- He who finds typos may keep them! ---

#3 george@CASE

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Posted 28 January 2005 - 12:51 PM

the ratio is molar ratio, not volume.

follow the formula given by jade falcon.




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