Ligation

ChenHA via methods%40net.bio.net (by hzhen from freeuk.com)
Fri Jun 29 20:10:05 EST 2007


Deitiker, Philip R wrote:

> My helper was concerned because the membrane clogged up on
> one sample and we were forced to discard some of the lysate. 

I don't know much about the Promega kit, so what I say may be 
irrelevant, but things can get clogged up by, say, precipated cell 
debris that wasn't properly removed, or cells not lysed and precipitated 
properly (can happens if too large a volume of cells are used, so just 
use proportionally more reagents in this case).


> 
> Typically I am retreaving 90ng/ul of DNA. 
> The vector is 6300 bp so this should give about 2.5 fm/ul
> [Who wanted fremptomoles? :^) ]
>

Seems good enough.  You don't need a huge amount of DNA for ligation, in 
fact, too high a concentration of DNA is bad for ligation - they tend to 
form concatemers rather than circularised plasmids.


More information about the Methods mailing list