is there a restriction about amount of vector in a ligation? - (Jun/03/2006 )

hello there,
I am wondering if anyone of you had before prepared a ligation reaction using only a 10 or 20 ng of cut vector?
I always do my ligations with at least 50ng of cut vector but this time I don't have enough of the insert to ligate a 50ng vector.
any suggestions will be appreciated
dodos,

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The concentration of DNA (vector or insert) has a strong effect on the number of concatamers in the final product. Plasmids with multiple origins usually do not replicate well, but are definitely formed in large numbers during ligation reactions. In our experience, low DNA concentrations during ligations are better than high concentrations. I would definitely try your ligation and expect good results. Concentrating the DNA following ligation is an option (precipitation or n-butanol water extraction). Do not try to make up for low DNA concentrations by adding more ligation mix to chemically competent cells. More than 5% is inhibitory in our experience.

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I have used 20ng of cut vector for ligation and has worked nice for some of my ligations. give it a try it should work.

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yes try it with 20 ng I got answer with 15 ng too

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In many hundreds (perhaps thousands) of successful cloning reactions, I have never checked the DNA concentration of either my vector or my insert, so I've probably been all over the map with regards to concentration. I just guesstimate ratios from the gel I purified the fragments from.

This is standard practice in my lab, and so not only do I not check concentrations, but neither do any of my lab mates, past and present, and they've also had many, many successful clonings.

Empirically then, the importance of checking DNA concentrations is way overblown, IMHO.

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I've gotten ligations to work using as low as 20ng of vector, maybe less. And those were blunt ligations on top of that. I think the molar ratios are more critical than the actual mass of DNA you use.

I estimated my DNA concentration by running them on a gel and comparing the band intensities. This isn't the most accurate method, but I've found our spec is even less accurate for measuring fragments such as cut vectors and PCR products, in fact we've relabled it the "The Guestimator"

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So if 15-20ng is the lower limit...what is an upper limmit?? Is 100-200ng too much??
Also...does anyone know about a lower limit for amount of ligated product in transformation?? I know I can transform unmanipulated vector anywhere from 50-100ng...but what about a ligation...presumably there is a lot less of your desired molecule and so do you have to transform more???

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i have had a succesfull ligation of 300ng vector once.
i generally transform 10-15ng of vector

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For some vectors, I had to use 200-300 ng of vector for ligation.

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Then how much for the transformation aftwards???

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