I have a question regarding a failed transformation using Origami2(DE3) cells. I tried a bacterial transformation of a pET28a containing plasmid (Kanamycin resistant) into Origami2 (DE3) cells. I used these cells as the expression of my target gene failed in conventional BL21 cell (stuck in cell pellet after cell lysis).
I used the transformation protocol as mentioned in the instruction manual (Merck Millpore - https://www.merckmillipore.com/NL/en/product/Origami-2DE3-Competent-Cells-Novagen,EMD_BIO-71345#documentation), however did not see any colonies on the bacterial plate.
My bacterial plates are a week old made with fresh LB/Agar and Kanamycin, the DNA I used for the cloning was ordered from Genscript and is verified by sequencing. I however thought if I had to add any additional antibiotics to the LB/Agar plate instead of Kanamycin.
After reading the cell manual of the Origami2 cells I found that the cells have additional antibiotic resistances like Streptomycin and Tetracycline. DO I need to add these too to the LB/Agar? Or is it something else that I need to consider while doing the transformation?
Thank you in advance for your suggestions!
Transformations are usually straightforward so when they fail you need to make sure you do all the right controls. So either the cells are bad or the plasmid is bad. Did you also transform a control plasmid (maybe the vector without an insert) or something else that you are positive is good plasmid? or transform your plasmid into a different strain at the same time. You need to figure out which is the problem.
But lastly also make sure that the plates are correct, sometimes whoever is making plates just makes a stupid mistake without thinking (like adds ampicillin instead of kanamycin by accident).
Regarding the other antibiotics, the Str resistance marker is a chromosomal mutation and you do not need to add. The Tetracycline is on the F' episome which is usually very stable. You can periodically check to be sure it is not lost but you don't need to maintain selection all the time.
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