Hi Kristi,

I have also been using the pDM vectors for the last few years.  I have never had any issues digesting the vectors.  We use the Fermentas Fast Digest enzymes - BglII and SpeI with the 10x Green Buffer.  I have conducted the double digest for only 30 minutes followed by Antarctic Phosphatase treatment.  The only time I have problems with the resulting digested vectors was if I did a gel extraction.  At that time I could not clone into the vector.  When I went back to the above protocol I had no problem cloning into the vector.

Good luck! 

Stephen Burrows
Ph.D. Candidate
Department of Biology
Clark University
Cell: 508-942-1056

________________________________________
From: DICTY [[log in to unmask]] on behalf of Dan Dickinson [[log in to unmask]]
Sent: Thursday, August 02, 2012 1:14 PM
To: [log in to unmask]
Subject: Re: [DICTY] listserv question: cloning into pDM310

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
>