IOWA STATE UNIVERSITY | DEPARTMENT OF AGRONOMY | COLLEGE OF AGRICULTURE AND LIFE SCIENCES | PLANT SCIENCES INSTITUTE

Submit your constructs to PTF

b. Submit Agrobacterium strains

Obtain APHIS interstate movement permit
1. An APHIS permit is required for transportation of Agrobacterium strains across state borders. It can take up to 120 days to receive approval of your application, so apply as soon as possible. Please go to the APHIS permit website for the application and detailed information http://www.aphis.usda.gov/permits/brs_epermits.shtml.
2. Once you have received approval from the APHIS department, please mail the permit number and a copy of the approval letter to Kan Wang (kanwang@iastate.edu) so we will be notified by the arrival of your strains.
Confirm DNA construct in Agrobacterium strain
1. Once you have introduced your construct DNA into Agrobacterium strain, please perform plasmid DNA mini-preparation from Agrobacterium culture.
2. Purify your plasmid DNA using a Qiagen plasmid DNA preparation kit. Note that you may need to start a larger Agrobacerium culture (compared with your typical DNA mini-preparation from E. coli) because plasmid DNA in Agrobacterium strain is usually of low copy numbers.
3. Confirm integrity plasmid DNA isolated from Agro strain with same plasmid DNA isolated from E. coli using electrophoresis by including
  1. undigested DNA
  2. DNA (both DNA isolated from Agrobacterium strain and from E. coli) digested with 2-3 restriction enzymes to confirm the construct.
    
    Restriction digestion
Mail strains
1. Ship Agrobacterium strains to PTF via overnight express (ice is not needed). The package should include:
  1. Stab culture or plate of bacteria, packaged as indicated in #2 in Mail Strains.
  2. Brief physical map of the construct with
    1. major restriction enzymes positions
    2. size of the construct
    3. antibiotics for bacterial selection
    4. selectable marker for plant, if applicable
  3. Gel picture illustrating the DNA quality (as described in #3 in Confirm DNA construct).
  4. Title and brief description of your project.
  5. Service requests include
    1. number of events desired
    2. type of product (seed, plant, callus)
    3. if request maize seeds, our standard pollen donors are B73 or Hi II for Hi II transgenic plants and B104 for inbred B104 transgenic plants. Alternative pollen donors may be possible if our greenhouse space is available. You will need to provide you alternative donor seeds.
  6. Your complete contact information including
    1. the name of PI, office phone, fax, e-mail (for billing)
    2. the name of contact person (if not PI), lab/office phone, e-mail (for construct communications and receiving samples)
    3. full mailing address including street numbers, if applicable
    4. FedEx number for shipping plant materials back to your lab
2. Agrobacterium strains must be packaged as follows:
  1. Stain must be in a primary inner container (stab culture or plate)
  2. Secure lid with parafilm (see below)
  3. Place tube or plate in a sealable secondary container (plastic bag)
  4. Place paper towel in the bag and seal (see below)
  5. Place bag in a cushioned envelope or box (see below)
  6. Enclose an itemized list of package contents
  7. Seal and mail
3. You will be notified by an e-mail message of the Identification Number (ID#) that has been assigned to your construct. PTF personnel will thereafter communicate with you using this ID# rather than the original name of your construct.