ratios for ligation reactions

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SororSaudade
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ratios for ligation reactions

Post by SororSaudade » Mon Feb 12, 2007 1:10 pm

Hello!

I was wondering if it is possible to know the best vector:insert ratio to use in a ligation reaction, knowing the sizes of both fragments.
Is there any way to calculate it, or you just have to try different ratios until you get the right one?

Thanks :)

lkmcneil
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ligation ratio

Post by lkmcneil » Mon Feb 12, 2007 1:44 pm

The insert should be present at a molar ratio of 3 to every 1 plasmid. If your plasmid is about 3kb and your insert is about 1 kb, then just use equal masses of each; 250 ng vector and 250 ng insert.

SororSaudade
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Post by SororSaudade » Mon Feb 12, 2007 3:04 pm

well, i'm working with a 3,7kb vector and a 1,6kb insert and 1:1 and 1:3 don't seem to work. I've now tried 1:5 and this seems to work but wih very low efficiency.
I'm trying to ligate blunt ends... maybe that's why the ratios have to be higher..
It would be very helpful if it was possible to calculate the optimal ratio before starting this kind of work.

Thanks

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