I used LB medium and incubated plate at 280C for 2 days. I've got some colonies but I'm not sure whether it is LBA4404 strain or not
Hi Nguyen,
1. It is generally hard to tell whether the colonies are LBA4404 just by reading the colonies' appearances.
2. You can use antibiotic selection to test it:
LBA4404: rifampicin and streptomycin.
Streak your bacteria on LB + rifampicin + streptomycin. The LBA4404 colonies should survive and form.
Also, you can use a pair of specific primers to amplify a unique fragment from LBA4404.
See this 2014 paper, on page 4 (left-handed lower corner). A pair of primers can be used to identify LBA4404. The amplified band is unique to LBA4404 and can be used to distinguish against other Agrobacterium strains (see attachment).
Hi Yuan-Yeu!
Thank you so much for your advices, it is really helpful for me
Hi Nguyen,
You are welcome!
By the way, I have observed, in general, the colonies of LBA4404 are much smaller in 2-3 days of growth, compared to for example E.coli colonies, if I streak them from -80C stock. Are your colonies tiny after 2-3 days of growth? But I still trust more on the results from the 'antibiotic selection' and 'molecular' methods I suggested above.
Hi Linh,
I agree with the suggestion from Yuan-Yeu. Try to grow LBA4404 on LB plate containing rifampicin and streptomycin. Normally, colonies of A. tumefaciens are quite small after 48 hrs, at 28oC. After 7-10 d, they look smooth, slightly viscous.
Enclosed you find a picture for typical colonies of A. tumefaciens AGL1. If you need a specific primer pair for A. tumefaciens, contact me via email: [email protected]
Best,
Tuan Tran
Thanks for your suggestion Yuan-Yeu, I streaked LBA 4404 on LB plate containing rifampicin and saw some colonies after 2 days. They're white, a little bit yellow and smaller than E. coli colonies. But after one more day, they're much bigger and look smoothy. I haven't try molecular method yet because I've not have the specific primer for A. tumefaciens.
Hi Tran,
Colonies that I got look similar to some on your picture. And I've already ordered the specific primer for selection. Thanks for your kindness!
Hi Nguyen,
Thanks for your update!
1. From your description, it is pretty much like the way I saw on my Agrobacterium LBA4404 growing on a plate.
2. I saw you only used one antibiotic for selection agent, it will be better if both of the antibiotics are tested for selection. This will make sure that some components in the cells are not lost.
3. Please update us regarding your PCR amplification when you get a result.
Normally, how long u guys grow the Agrobact in broth (LB + AB : in my case LB + Chloramp as my plasmid carries the resistant gene) to obtain OD at 600 of 0.8 to 1.0. Mine overnight. Is that normal ? As some paper mentioned it takes 2 days minimum to obtain the nice concentration. Thank you!
you may confirm Agrobacterium with Benedict’s Reagent ,
https://www.ars.usda.gov/ARSUserFiles/20300515/PotatoMolecularGenetics/pdf/Benedict.pdf
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning