Dear all, i was making a binary vector for plant transformation. It had spectinomycin gene in the vector with pPZP backbone, I replaced this with kanamycin gene. its is working with E.coli strain (DH10B which we are using in our lab) with 50 µg/ml kan. concentration but not with Agrobecterium strain (GV3101) with the same concentration . Before this we made an other vector with kanamycin gene using the same PZP backbone which was working on 50 µg/ml kan. concentration but this vector is working on 30 kanamycin conc. for agrobacterum and the bacteria are growing very slowly. Kindly help me to solve the problem.
Hello Walter,
Thanks for suggestion, now i am exactly trying the same thing by inverting the orientation of the gene and i hope it will work. I also sequenced the kanamycin gene and there was only one mismatch in protein (point mutation but not frame shift) but that was not on active site of the gene. What do you think this problem might be due to this mutation.
Regards
Hi Walter,
Thanks for reply, I am sure you will be succeeded in the interview hopefully. yes i just changed the orientation of the gene as suggested by you and it is now working on the prescribed kanamycin conc. Thanks again for reply and nice suggestions.
Wishes
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