I have Agro (GV3101 competent cells) with my plasmid that I want to isolate. I miniprepped from Agro using a plasmid DNA isolation kit, which yielded approximately 25-35 ng/ul plasmid. I tried to transform E. coli (NEB 5 alpha competent cells) using the manufacturers protocol, something thats always worked for me (heat shock with 50-100 ng plasmid, 1 hour incubation in SOC outgrowth medium, then plating on LB Kan selection, then incubation overnight at 37 degrees). However, I cannot get colonies to grow. My positive control transformation worked. I've already tried using both 50 and 100 ng plasmid for transformation, and plating both low and higher concentrations of the SOC outgrowth culture on the plates. Does anyone have any troubleshooting advice?
Thanks!
have you checked if this is a shuttle plasmid ... remember that e.coli a agrobacterium are different and you need different origin of replication for each host ... its not going to work if your plasmid is only At plasmid. of course proper selection and the size are a key too as Pieter told you if you have pTi plasmid its about 200kb and never going to work with chemical transformation even if it has both origins and Km selection... try elecroporation or conjugation if its designed for it.....
Hi April
It seems like the problem is in the plasmid, as the pos. control transformation worked. I assume that your plasmid has a kanamycin resistance gene (and that the resistance is not property of the original GV3101 strain)?
What does your plasmid look like on an agarose gel? Is it the size you expect? Pure? You can try sequencing the kanamycin-resistance region/bacterial ori to confirm that it is intact and can confer resistance.
What is the size of your plasmid? Large plasmids (>10kb) may be more difficult to transform and require more slowly growing competent cells, such as DH10B.
Also I assume you are using chemically competent NEB5 alpha cells and not electrocompetent ones?
I hope any of these suggestions can point you in the right direction.
have you checked if this is a shuttle plasmid ... remember that e.coli a agrobacterium are different and you need different origin of replication for each host ... its not going to work if your plasmid is only At plasmid. of course proper selection and the size are a key too as Pieter told you if you have pTi plasmid its about 200kb and never going to work with chemical transformation even if it has both origins and Km selection... try elecroporation or conjugation if its designed for it.....
it sounds that the problem is related to the plasmid not the competent E.coli, you can check if the plasmid contains other resistant gene than kanamycin and try it, also, you can try the heat shock for longer time to 45 seconds, hope it helps !
Hi, April DeMell.
Could you tell me more about your plasmid? It's really difficult to give you any point of view without all data. So, what plasmid are you using? What is it size and the is the size of your gene of interest? Is it for cloning or expressing a protein? Did you check the plasmid by doing some sequencing or RE digestion to confirm it is really what you're looking for?
First you can check for the presence of the origin of replication (ori) of E. coli within Agro plasmid.
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