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1. What is the selection agent used for plant transformation in Creative Biogene?
There are many available selection agents, such as Hygromycin, G418, basta, glyphosate, mannose.
2. How much plasmid should be sent to you?
For Agrobacterium transformation, 800 ng is needed. Plasmid DNA should be from a high-quality miniprep (intact DNA with ratios in the range of 1.8 to 2.0 at OD260/OD280).
For biolistic transformation and PEG-mediated protoplast transformation, 10 ug of each plasmid diluted in sterile water at 1 ug/ul is needed. A poor quality DNA sample will lead to a poor quality transformation, so we suggest that it should be prepared using a plasmid DNA maxiprep kit.
3. What Agrobacterium strains are used for plant transformation in Creative Biogene?
The Creative Biogene's protocols were optimized using Agrobacterium strains EHA101 for maize, tobacco and soybean, EHA105 for rice and Arabidopsis, AGL-1 for wheat, barley, and rice, GV3101 for potato and cassava, LBA4404 for tomato and cotton.
4. Can you use my Agrobacterium strain?
Yes, other strains can be used. But this may result in less than optimal transformation results, so 10 independent events cannot be guaranteed.
5. Will you provide binary vectors and Agrobacterium strains for gene construction and Agrobacterium transformation?
Yes, we provide gene construction service including overexpression vector, silence vector, editing vector and complementary vector. All of our modified Agrobacterium strains can be sold to customers.
6. How many plants do you provide for each construct?
We provide a minimum of 10 independent events per construct. In the case of Arabidopsis, we provide the T1 seed of at least 10 selected transgenic events.
7. How long will it take to generate transgenic plantlets or seed?
It usually takes 6 to 10 months to get transgenic lines. Crop plant transformation is a difficult process, in general, the turnaround time mainly depends on the construct and how it interacts with the plant cells.
8. What method do you use to characterize the transgenic plants?
We generally run a PCR-based assay to detect selectable marker and ensure the accuracy of detection result at genome level.
9. Do you accept constructs containing gene editing technology?
Yes, we offer customized gene editing service. In generally, the selection of sgRNA and construct is a crucial factor to editing efficiency. For this reason, minimal positive event number may not be guaranteed. But we will try our best to deliver independent transgenic events as many as possible. In addition, we also provide comprehensive genome editing service, covering from sgRNA design, vector construction, transient validation in protoplast, genetic transformation to mutation detection.
10. Can you test my plants for the presence of my transgene or gene edit?
Yes, we offer qPCR and WB analysis to investigate the presence of transgene. For genome editing, a project report will be delivered to customers, which includes the results of target site sequencing, PCR-RE assay and deep amplicon sequencing.
11. What are the differences between biolistic transformation and Agrobacterium transformation for plant and which one to choose?
Agrobacterium transformation is the most common method which shows advantages like low copy numbers integration, less expensive, high efficiency, stable integration, minimal DNA rearrangements. When using binary vectors, it is recommended to transform with Agrobacterium.
Compared with Agrobacterium transformation, biolistic transformation is more efficient and has wider applicability. The vectors used in biolistic system can be relatively small and easier to work with. In addition, biolistic transformation may be advantageous for genome editing to obtain transgene-free events.
12. How does the Creative Biogene define independent events?
The Creative Biogene is very conservative about what is called an independent event. Independent events are counted only if plantlets come from different transformed explants. For example, if two plants are regenerated from the same position in a single rice callus, then we count these as clonal events.
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