Reasoning behind optimum insert:vector ratios?

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Reasoning behind optimum insert:vector ratios?

Post by Katianna » Tue Oct 19, 2010 10:57 am

Dear all,

I am working with bacteriophage vectors, but I know that vector/insert numbers are similar in other vectors. In my protocol there is call for the use of insert:vector ratios of 1:1 to 3:1, though I know people have gone to higher ratios.

My question is what is the reasoning behind having more insert? Is it to do with the expected quality of prepared insert? Something to do with the vector? I can't work out the reason. Any help is hugely appreciated.

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Post by JackBean » Tue Oct 19, 2010 11:22 am

the reason is, that you want the insert to be incorporated into the vector, while it can be self-ligated being empty as well. In such a case, you will get colony, because it has working plasmid, but there is no insert. While if you increase the amount of insert, there is higher chance it gots incorporated.

Cis or trans? That's what matters.

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