Ligation

newsnet customer via methods%40net.bio.net (by customer from newsnet.com)
Mon Oct 16 06:43:27 EST 2006


Hi,

Sambrook recommends DNA as low as 10ng for ligation. The low concentration is required for DNA to circularise. I was just wondering if this concentration is for the vector or insert or both? 

I read on a website that the vector should be 100ng and the insert should be anything from 10ng to 100ng (depending on the molar ratio and vector to insert size).

The site also recommends trying different insert concentrations, to find out what works best.

vector 100ng + 10ng insert
vector 100ng + 20ng insert
vector 100ng + 50ng insert
vector 100ng + 100ng insert

It seems like the vector concentration is fixed at 100ng, while the insert can vary from 10ng. What are peoples thought?

Here is the website: http://www.soton.ac.uk/~kpa/molecol/clone.html

Cheers,
ST


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