Hi Kristi, 

I did a lot of cloning in pDM310 while I was in grad school.  I never had much trouble.  It is important to use good competent cells (I used XL10 gold) because the vector is big.  I routinely used Bgl and Spe together in NEB4 buffer for 1 hour.  Since Bgl is supplied with NEB3, I use twice the normal amount of Bgl (20U instead of 10).  

I suggest that you test your enzymes individually to make sure they each cut before using them together in a double digest.  

Good luck!
Dan Dickinson


On Aug 2, 2012, at 11:56 AM, Kristi Miller <[log in to unmask]> wrote:

> Dear fellow Dicty enthusiasts,
>  
> I am currently trying to subclone a gene (1200 bp) into the pDM310 dox-on inducible vector (8471 bp). Does anyone who has worked with this vector previously have any reccomendations as to how long the vector should be digested with SpeI and BglII before gel extraction, ligations, and transformations? I have tried double digests for 45 minutes, sequential digests of 45 minutes each as well as sequential digests for 15 minutes each.
>  
> Has anyone else had trouble cloning into the pDM310 inducible vector? Any suggestions on how to overcome this problem?
>  
> Thanks!
> Kristi
>  
>  
>  
> Kristi Miller
> Master's Candidate
> Graduate Research Assistant
> Central Michigan University
> Dow Hall 246
>