Acquire Plasmids

The process of submitting requests to the Gene Therapy core lab will require both completion of information on the web site, as well as the submission of necessary starting materials such as the plasmid DNA and the relevant information. There are two different types of plasmid submissions which we receive:  1) submissions of plasmids that can be used directly to produce virus by transfection (“virus-ready” plasmids), and 2) submission of plasmids that can be used as a source of the genes to be subcloned into viral vectors.  The requirements for the information we need are different for each type of plasmids.  Note that you may submit a plasmid which does contain sequences for the production of virus as just the source of a gene.  (e.g., "Please move this gene from our retrovirus plasmid into an AAV vector.")

 

To submit a “virus-ready” plasmid, please provide the following:

1)  At least 35 micrograms of purified DNA

2)      A completed form for plasmid DNA submission to HGTI via our website.

3)      A restriction map of the plasmid, showing the relevant features and restriction sites.  Please include an image of a gel showing at least two diagnostic restriction digests (For AAV vectors, a SmaI digestion should be included).

4)      An electronic DNA sequence submitted via our website which represents (preferably) the entire plasmid DNA you are submitting.  If you do not have access to the complete sequence of the viral vector DNA, please provide us the sequence of the inserted fragment.

 

To submit a plasmid as the source of a gene, please supply the following:

 

1)  At least 35 micrograms of purified DNA

2)  A completed form for plasmid DNA submission to HGTI via our website.

3)  A restriction map of the plasmid, showing the relevant features and restriction sites.  On the     map, you will need to indicate your preference for the restriction enzymes that we will use to drop out the gene and insert it into the vector plasmid.  We will be linkering the fragment in order to insert it into the vector, and so you do not need to worry about overhang compatibility.  We would recommend you chose the enzymes that produce the smallest fragment that contains the full open reading frame of your gene and Kozak translation initiation sequences.

Please include an image of a gel showing at least two diagnostic restriction digests.  The gel must include a lane where the DNA is cut with the enzyme(s) that will drop out the fragment for subcloning.  The fragment ought to be reasonably separated from all other bands.

4) An electronic DNA sequence submitted via our website.  The electronic DNA sequence must consist of at least the fragment that we will be purifying and inserting into our vector, including the sequence of the restriction site or sites that we will be using to liberate the gene.  This obviously includes the full sequence of the gene.

Within the electronic sequence, you will need to provide the exact numeric coordinates that correspond to the start and stop codons of the open reading frame that you intend to move into the vector.