Hi Everyone. I am planning to do agrobacterium transformation after LR reaction. I use EHA105 agro strain for transformation. the concentration of one of my vectors (PGWB661) is 70.792 ng/µl. I use 1.5 µl of this vector for 50 µl agrobacterium competent cells (about 100 ng/µl). I used 2 different methods, heat and shock and electroporation for transformation. after 3 days I didnt get any colonies. I know that heat and shock method needs more time for colonies to grow, but electroporation method should answered. Do you have any suggestions?
Hi Mahboubeh,
how did you prepare your competent EHA105 and did they work with other vectors before?
I am just asking because we found that, for us, the Ca-based protocol of An et al. (1988) did not work well. We switched to the protocol of Höfgen and Willmitzer (1988; doi.org/10.1093/nar/16.20.9877) which works very well in our hands.
The size of your vector is in the same range as the pBIN and CAMBIA series of vectors, with which we had no problems. pGWB661 seems to harbor an aadA gene. What antibiotic did you use for selection? We usually use both, spectinomycin and streptomycin, at 50 mg/l. But, in our work we did not use Spec/Strep based selection for EHA105. It seems that the sensitivity to the antibiotics varies quite a bit between agro strains. For instance, for LBA4404 we had to lower the concentration of streptomycin to 30 mg/l (instead of the mentioned 50 mg/l) to get any transformants. Maybe you have to test that for your EHA105 strain, too?
Good luck!
Regards,
Klaus
I did this wiith agronacterium tumefaciens. The recommendation is that to get fresh strain (bacteria), it is neccesary to isolate from the tissue, I mean from the tumors you have to get some explants in order to isolate in the agrobacterium.
Mina Bayanati
Hi Mina. thank you very much for your reply. Actually the Agrobacterium strain is ok, because I used it for another construct and It was ok.
Klaus Eimert
Hi Klaus,
thank you very much for your explanation. I use Kanamycin, Rifampicin and Spectinomycin (each 50 mg/l) for selection and I use YEB medium. I agree with you. I use the same method as mentioned in your reference just instead of 5 minutes in ice, I put 30 min in ice, then 5 min in liquid nitrogen and 5 min at 37C. Adding 500 μl S.O.C medium and incubating at 30C for 2-4 hours. after centrifuge, I cultivate 200 μl in each plate.
Hi Mahboubeh,
for the transformation protocol - a critical point in our hands is the heat shock at 37°C. The agros seem to be very sensitive. Thus, we are very careful not to exceed the 5 minutes.
As for the antibiotics, you included Kan in your list. If I remember correctly, the original pGWB661 vector does not have a NPT gene, neither for prokaryotic nor for plant selection. Did you modify it?
Regards,
Klaus
Klaus Eimert no. I didn't modify. Sorry maybe I explained in a wring way. For pGWB461 I use Rif and Kan. For pGWB661 I use Rif and Spec. About the temperature, I don't exceed more than 5 min at 37C.
Hi Mahboubeh,
OK, I see. Do you have this problem with both vectors? Did you get transformants in EHA105 with other vectors (other antibiotic selections)?
I am still not sure I understand you right. In pGWB461 prokaryotic selection with Kan should not work. The NPTII (in the T-DNA) is under NOS-promoter control which is supposed to work in eucaryotes only. The bacterial selection should also be based on the aadA gene (specR and strepR) as in the pGWB661 vector. Am I missing something?
Cheers,
Klaus
Klaus Eimert
Hi Klaus,
I got colonies for pGWB461 with kanamycin selection antibiotic. I did colony pcr, as well, but for pGWB661, no. I repeated again with some changes in my protocol. I hope I could get colonies.
Klaus Eimert Dear my friend,
I could get good colonies for my constructs. I changed my protocol and used the protocol that you mentioned in your previous email. I decreased the incubation time in ice from 30 min to 5 min and it was really good. after 3 days, I got good colonies with where correct after colony PCR. thank you very much for your instructions.
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