Ordering Procedures

Choose a binary vector  | Request a vector  | Submit constructs to CBL | Submit project request form | Apply for shipping permits | Receiving plantlets

Choose a binary vector 

There are two different types of binary vectors available for you to choose from: 

A. pCBL101:

Good for complementation studies, over-expression, etc. using your own expression cassette (promoter + gene + terminator). This vector does not contain a promoter to drive the expression of the gene of interest.

Basic features of the vector:

• A broad host range (pVS1) origin of replication.
• An SmR gene for bacterial selection of spectinomycin resistance.
• The T-DNA right and left border fragments from a nopaline strain of A. tumefaciens.
• A selectable marker gene cassette containing (1) ZmUbi promoter, (2) NptII coding region, (3) PINII terminator.
• A multiple cloning site containing nine unique restriction enzyme sites (Eco53kI, SacI, Acc65I, KpnI, TspMI/XmaI, SmaI, SbfI, BspEI).

Other pCBL101 versions:

pCBL101.1 carries a different set of restriction enzyme recognition sites at MCS (Eco53kI, SacI, AscI, AvrII, Acc65I, KpnI, MluI, SacII, SbfI, BspEI)

pCBL101gw is a Gateway one-entry version that contains attR1 and attR2 recombination system suitable for subcloning your Gateway compatible gene of interest cassette (promoter + gene + terminator)

pCBL101gg is a recipient vector for 4-way assembly via Golden Gate cloning. After cloning the promoter to pCBLggPro, coding sequence to pCBLggCDS, and terminator to pCBLggTerm, one can assemble them with pCBL101gg in a single tube using T4 DNA ligase, BsaI, and a thermocycler. 

B. pKL2351 

If you are thinking about using CRISPR technology to edit your gene of interest. pKL2351 is your choice.

Basic features of the vector:

• A broad host range (pVS1) origin of replication
• A Kanamycin resistant gene for bacterial selection of kanamycin resistance
• The T-DNA right and left border fragments from nopaline strain of A. tumefaciens
• A selectable marker gene cassette containing (1) ZmUbi promoter, (2) NptII coding region, (3) PINII terminator.
• A Gateway entry system contains attR1 and attR2 recombination sites

Depending on the number of sgRNA you want to insert, you may want to use different entry vectors to build the final binary construct. See below for entry vector information.

 Entry vectors for making CRISPR construct using pKL2351 as destination vector. Contact Dr. Jessica Ji for cloning more than 2 sgRNAs. 

 Request a vector

All above mentioned vectors are available from Addgene now. For special vectors that are not available from Addgene, please follow the steps below to request these vectors directly from CBL:

• Contact us for the standard Material Transfer Agreement (MTA).
• Complete the MTA by providing the required information and obtaining signatures of both the Principle Investigator (PI) and the institutional authorized officer.
• Send your request, the signed MTA, and your FedEx account number by email to Kan Wang (kanwang@iastate.edu) and Jessica (jjessica@iastate.edu), including the full mailing address and specific request on the desired vector.
• Send an acknowledgment email to Kan Wang (kanwang@iastate.edu) and Jessica Ji (jjessica@iastate.edu) upon receiving the vector DNA.

Submit your constructs to CBL

Submit DNA constructs

You can submit your constructs as Agro glycerol stocks (internal customers only) or as plasmid DNA. If you submit your DNA constructs, we will introduce them into appropriate Agro strains before plant transformation. For details and prices, see Services and Fees.

Preparing DNA

• Purify your plasmid DNA using a Qiagen plasmid DNA preparation kit. Dissolve DNA in ddH₂O, not TE!

• Measure DNA concentration using a spectrophotometer. Obtain OD₂₆₀ and OD₂₈₀ readings. Dilute DNA to 250 – 1000 ng/μl. Acceptable DNA purity is within an OD₂₆₀/OD₂₈₀ range of 1.6 to 1.9.

• Check DNA quality (500ng/lane) using electrophoresis by including: a. undigested DNA; b. DNA digested with 2-3 restriction enzymes to confirm the integrity of the construct.

Mail DNA

1. Ship DNA to CBL via overnight express (ice is not needed). The package should include:

• 5-10 μg DNA in ddH₂O (concentration of 250 – 500 ng/μl), packaged as indicated below in #2 in Mail DNA.

• OD₂₆₀ and OD₂₈₀ values and ratio

• Gel picture illustrating the DNA quality as described above in 'Preparing DNA' section. Alternatively, you can also email CBL your gel picture.

• Map of the construct with 1) major restriction enzymes positions; 2) size of the construct; 3) antibiotics for bacterial selection; 4) selectable marker for the plant. Alternatively, you can email CBL the SnapGene format of your construct.

2. Pack your plasmid DNA as follows:

• Plasmid DNA must be in a primary inner container (1.5 ml tube)

• Secure tube lid with parafilm

• Place the tube in a sealable secondary container (plastic bag)

• Place a towel or Kimwipe inside and seal the bag

• Place the bag in a cushioned envelope or box

• Enclose an itemized list of package contents

• Seal and mail, our mailing address is:

Jessica Ji
G0401 Agronomy Hall
716 Farm House Ln
Ames IA, 50011

Submit your project request form

Fill out the project request form and obtain the signatures of both the user and the Principle Investigator (PI) and send it back to Jessica Ji (jjessica@iastate.edu).

For cloning service, please submit the cloning service request form.

Upon receiving all required materials from you, CBL will assign an Identification Number (ID#) to your construct and you will be notified by an email message. CBL will thereafter communicate with you using this ID# rather than the original name of your construct.

Apply for shipping permits

APHIS BRS interstate movement permit
Introduction (importation, interstate movement, or environmental release) of transgenic materials are regulated by APHIS Biotechnology Regulatory Services permits. The details of the regulation can be found on APHIS website https://www.aphis.usda.gov/aphis/ourfocus/biotechnology/regulatory-processes/permits/permits.

Apply for Permit
Detailed guidance for permit application can be found on this APHIS site: https://efile.aphis.usda.gov/s/brs-permitting-assistant. You need to submit a Permit application via APHIS eFile on APHIS website. Because it can take some time to obtain your Permits, it is recommended to start the application as soon as you submit your construct for transformation. Transgenic corn plantlets can be ready to ship in 8-9 weeks after infection on average.

For questions regarding APHIS permits, send inquiries to biotechquery@aphis.usda.gov. 

Receiving plantlets

Shipment of transgenic plantlets will be scheduled with you ahead of time. You will be notified by e-mail 1-2 weeks before plantlets are ready to be sent. You may receive shipments of plantlets over several different dates because plantlets develop at different rates. Remember that the plantlets cannot be shipped without your APHIS interstate movement permit.

To grow transgenic maize plants to maturity in your own greenhouse, keep in mind that you will need to plan for planting tester lines. These tester lines will serve as your pollen donor plants for transgenic silks, or silk recipient plants for transgenic pollen, since it is usually difficult to have a transgenic plant to produce its silks and tassel at the same time. 

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