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

Submit your constructs to PTF

a. Submit DNA constructs

You can submit your constructs as plasmid DNA for both Agrobacterium-mediated and biolistic-mediated methods. Plasmid DNA submitted for the biolistic transformation should be of high quality (see below for DNA preparation guidelines). If you submit your Agro constructs, we will introduce them into appropriate Agro strains before plant transformation. For details and prices, see DNA-Agrobacterium transformation service.

Preparing DNA
1. Purify your plasmid DNA using a Qiagen plasmid DNA preparation kit. Dissolve DNA in ddH₂O, not TE!
2. Measure DNA concentration using a spectrophotometer. Obtain OD₂₆₀ and OD₂₈₀ readings. Dilute DNA to 0.5 – 1 µg/µl concentration. An acceptable DNA purity is within an OD₂₆₀/OD₂₈₀ range of 1.6 to 1.9.
3. Check DNA quality (1 µg/lane) using electrophoresis by including
  1. undigested DNA
  2. DNA digested with 2-3 restriction enzymes to confirm the construct.
Mail DNA
1. Ship DNA to PTF via overnight express (ice is not needed). The package should include:
  1. 5-20 µg DNA in ddH₂O (concentration of 0.5 – 1 µg/µl), packaged as indicated in #2 in Mail DNA.
  2. Construct map
    
    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. OD₂₆₀ and OD₂₈₀ values and ratio
  4. Gel picture illustrating the DNA quality (as described in #3 in Prepare DNA).
    
    Gel picture
  5. Title and brief description of your project.
  6. 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.
  7. 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. Plasmid DNA must be packaged as follows:
  1. Plasmid DNA must be in a primary inner container (1.5 ml tube)
  2. Secure tube lid with parafilm (see below)
  3. Place tube in a sealable secondary container (plastic bag)
  4. Place a towel or kimwipe 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.