minimum time for drop dialysis?

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Wed Jul 26 10:15:04 EST 2000


We never dialyse before ligation, so the minimal time is zero. In fact,
ligase works well in medium salt buffers (supplemented with 1mM ATP, of
course!). Anyway, you can simply dilute the digest; we use to digest 1ug
of DNA in 10ul, so we can use 1ul (=100ng) in a standard 10ul ligation
mix (containing ligation buffer usually supplied with the ligase). That
is a 1:10 dilution and you really don´t have to care about the
restriction buffer composition. 

Hope this helps,
Frank


Richard Converse wrote:
> 
> Dear archive:
> 
>    I had a simple question:
> 
> What is the minimum amount of time for filter dialysis of a restriction
> enzyme digestion in order to resuspend it in ligation buffer for an
> overnight ligation?
> 
> I know that for electroporation, a 25ul rxn is good to go for zapping in 15
> min. against 5ml of TE--is this long enough for ligations to work well?
> 
>   e-mail any responses to converrl at hotmail.com...
> 
>                  Many thanks,
> 
>                  Richard--
> ________________________________________________________________________
> Get Your Private, Free E-mail from MSN Hotmail at http://www.hotmail.com
> 
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