I isolated my plasmid (includes my insert) from GV3101 by mini prep kit but the concentration is too low. is there any other method to isolate the plasmid from GV3101?
What are you doing with your isolated plasmid? Are you checking it for your insert? Agrobacteria grows slow and doesn't make a lot of plasmid in the first place. You can either use a much larger volume of culture in your prep (do a midiprep instead of a miniprep), or you can use your current GV3101 plasmid to re-transform E. coli. You should have enough plasmid from your Agro prep to do this. If your E. coli carries the proper construct, then your GV3101 clone is most likely good. Since E. coli will make a ton of plasmid, you should get good yield and be able to test your construct and continue on with your Agro clones.
what is your point? you mean the concentration is too low, the concentrate is the plasmid or the GV3101? If your GV3101 is cultured in culture, the concentrate will be higher?
What are you doing with your isolated plasmid? Are you checking it for your insert? Agrobacteria grows slow and doesn't make a lot of plasmid in the first place. You can either use a much larger volume of culture in your prep (do a midiprep instead of a miniprep), or you can use your current GV3101 plasmid to re-transform E. coli. You should have enough plasmid from your Agro prep to do this. If your E. coli carries the proper construct, then your GV3101 clone is most likely good. Since E. coli will make a ton of plasmid, you should get good yield and be able to test your construct and continue on with your Agro clones.
I agree with Emily, retransform the plasmid DNA from A.tum to E.coli. The yield from A.tum is very low and you can only see bands >1kbp in a digest using half your plasmid amount. I now routinely perform colony PCR on the A.tum after the transformation and use at least 2 primer pairs that cover the T-DNA region of the binary plasmid to make sure the plasmid is intact.
The main reason for getting low yield of plasmid DNA isolated from Agrobacterium no matter which strain you typically use is due to the fact that the the plasmids used for plant transformation nearly all have origin of replications allowing only low plasmid copy numbers. Most plasmids we use in resaerch are optimised for high copy replication in E. coli and this is not done for Agrobacterium yet. So most plasmids in E. coli can be present between 100 to 500 copies but this number for T-DNA plasmids are about 10 copies. Therefore, the method of plasmid isolation will only minorly influence the yield optained. As suggested by Emily, you should either increase the culture volume therefore the amount of bacteria used for DNA isolation or re-transform the plasmid you isolated from Agrobacterium into E. coli.
We started-up a company called AgroJector and this issue will be one of the improvements we will try to make by genetically modifiying the Agrobacterium strains and developing better plasmids. In case you are interested in our company here is the link.
www.agrojector.com
I agree with the others that the best method is to perform your usual miniprep with a culture of agrobacterium. Due to the low copy number and general poor efficiency of minipreps with agro, you should take your agro miniprep and transform it into E. coli. You might even want to increase transformation efficiency into E. coli by using electroporation instead of heat-shock. Don't worry if you nanodrop the agro miniprep as you wouldn't expect to see anything due to the low concentration. This method has worked for me in the past when I wanted to extract a plasmid from an agro strain that another lab sent me.
thank you all for your kind replies. I had plasmid with my gene of interest transformed in E.Coli. my main concern is checking the desired plasmid in the Agro GV3101 before infiltration to the Nicotiana benthamiana.
my competent cells are growing within overnight incubation though transformation is Ok but I am worried that transformed have my desired plasmid or not.
thanks ,
Kind Regards
Pratima
If you want to know that you have the correct plasmid in your agro strains just use PCR. Use a vector-specific primer for one direction and an insert-specific primer for the opposite direction. If you are using a 50 uL reaction, just add 1 uL of your O/N agro culture as template. I usually get pretty clear results with this method but I wouldn't recommend using more than 1 uL of culture in a 50 uL PCR reaction as this might mess up your reaction.
Why do you check the sequence of the plasmid to verify or part of the insert itself. Or just by pcr.
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