Hello!
So, I have my gene cloned in a PSAT silencing vector which has been validated through sequencing. i have the plasmid in hand and I am trying to transform it into Agrobacterium GV3101 strain using YEPA with RIF and Amp selection antibiotics. The problem that I'm facing is I am getting a lawn kind of formation on my plates every single time I proceed with the plating. I have also tried LBA plates but the results are the same. on streaking the lawn, I am not getting single colonies, and the very few that I get is being proceeded with colony PCR with a blank gel profile. both gene specific and vector specific primers are being used. Please offer suggestion! Thank you.
Make sure your antibiotics have not gone off by also platting untransformed cells to see that all are killed i slection process. If not make new plates, if still hetting a lawn, plate dilutions until single cell colonies present. If too many cells plated, some without resistance can survive within halo of positively expressing cells degrading antibiotics.
Best !
I agree with Charles, you are most likely having a technical problem either with the strain you are using or the media. Repeat with all the appropriate controls, such as no DNA, your test DNA, and if you have, a known positive DNA control.
Thank you kind Sir, I have tested my competent cells as well as the media. Both are working fine. I've transformed my cells yet again today decreasing the incubation temperature and shall follow up with the dilution aspect while plating.
I think if cells and antibiotics are Ok, serial dilution should work
Sir, serial dilutions haven't given me results. I'm still getting the same type of biofilm like lawn. I shall restart my transformation process with other Agrobacterium strains available in the lab soon. I also had huge issues trying to create the silencing construct in the first place. I'm also trying to analyse the gene for its potential mutagenic roles.
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