I have been trying to make this A. tumefaciens c58ci from last year glycerol stocks stored at -80C competent.I firstly grew in LB medium supplemented with rifampicin and gentamicin. One thing I noticed after harvesting the cells was that they were chocolate in colour; I was wondering if this was normal? After making the cells competent, I transformed them with plain PBI121, spread appropriately on plates supplemented with rifampicin and kanamycin. The plates were at incubated at 30c but could not get any colony after three days. Do you think I am doing something wrong? Or I still need to wait for few days. Please kindly advise.
Thank you.
If you have been growing for a couple of days, it is normal for your At culture to turn chocolate. please check the OD of your bacterial population to determine the concentration.Ideal OD at 600 will be 0.5-0.6 for any downstream application. I'm thinking your growth period might be way too long.
Thanks @Nisha, I have been grappling with making this agrobacterium competent for about a month now and it's really slowing me down. Pls can you give me more vital tips for making the cells competent and transforming them? I appreciate any useful tips and advice.
Just saw this. Probably you have figured out by this time. See the attached file for the protocol I used to follow for making agrobacterium competent cells.
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