Hi,
I would like to have a clarification about the optimization of the condition reactions in a ligation.Why a larger amount of cut vector is used if plasmid is larger and a larger amount of insert is used if this is larger? I expected the opposite..I expected that if a plasmid is bigger, I need to take less of it and the same for the insert. So, why not?With regards to this, I read on a book that for a vector of 5kb and insert of 500 bp you would use an amount of vector ten times that of the insert in a 1:1 ratio. Why is the vector supposed to be 10 times the insert? Shouldn't be ten times less? I also heard about that the ratio of insert and vector depends on the concentration of steaky ends of these two..Whta does it mean?
Thank you very much
When optimizing a ligation, it's important to think in terms of molar amounts of vector and insert rather than in terms of weight (although almost all molecular biology protocols refer DNA concentrations in weight terms unfortunately). I usually shoot for a 4:1 to 6:1 ratio of insert to vector (again a MOLAR ratio, not a weight ratio). So if my vector is 6kb and my insert is 1kb, I add equal ng amounts of DNA to obtain a 6:1 ratio. If the sizes were instead 6kb and 500bp, I'd add twice as much ng of vector as insert. If I added equal weights instead like I did in the former case, I'd be adding twice the molar equivalents of insert and end up with a 12:1 ratio instead of a 6:1 ratio. On a side note, I often find that the ratio doesn't really matter a ton for getting my insert into my vector...I usually just pick values that are close (the 6kb vector and 1kb insert are close approximations for most of my work) and go with it.
https://www.neb.com/tools-and-resources/usage-guidelines/tips-for-maximizing-ligation-efficiencies
http://bitesizebio.com/10203/ligation-optimization/
In a ligation, the two ends of plasmid ligate with two ends of the insert, so at least an equal number of insert and vector is required. The numbers are represented by molarity, which is taken into consideration in the calculation and asks for the length of plasmid and insert.
It's like if you want equal number of watermelon (plasmid) and apples (insert), watermelon will be required more by weight.
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