I am trying to purify a plasmid from agrobacterium using Alkaline Lysis method, but it seems very difficult to get it done. Has anybody tried to purify any plasmid from Agro? How? What is the best procedure for that? Does STET help better than Alkaline Lysis?
Please share any experience, and thank you all in advance.
Mustafa
Hi
I did Agrobacterium plasmid using thermoscientifc kit. it was not as we get from E.coli but it was good enough to have digestion. other wise u can use qiagen kit.
1. In general, Agrobacterium gives you low copy numbers of plasmids, not like miniprep from E. coli. For a miniprep, you can only harvest small amount of plasmids from Agrobacterium.
2. To isolate large quantity of plasmids-of-interest from Agrobacteria, you need to grow high amount of Agrobacterium culture (such as a few hundreds of mL liquid culture) and use a high-capacity column (such as Midi-, Maxi-, or Mega-preps) to isolate them if you choose to use commercial column kit.
Agrobacterium plasmids are low copy number plasmid hence sometimes it becomes difficult to isolate them by alkaline lysis method. So, you can try Qiagen plasmid isolation kit.
Hi,
Although my experience concurs (low copy), I was able to successfully harvest low-copy plasmids from 5mL of fresh culture:
https://www.researchgate.net/publication/27559965_Characterization_of_the_Cardiolipin_Synthase_from_Arabidopsis_thaliana p.51.
Article Characterization of the Cardiolipin Synthase from Arabidopsis thaliana
I appreciate all of your valuable answers. They are really helpful.
Mustafa
Hi Mustafa,
You could also try the following, it is very simple to follow
What you will need:
Lauria broth medium supplemented with appropriate antibiotics
Cell suspension solution: 50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA (pH 8.0)
20 mg/ml lysozyme solution
Cell lysis solution: 0.2 M NaOH, 1.0 % SDS
Phenol solution
Neutralization solution: 3 M Na-acetate (pH 5.2)
TE buffer: 10 mM Tris-HCl, 0.1 mM EDTA (pH 7.8)
Agrobacterium tumefaciens plasmid isolation
The bacterial culture was inoculated in Lauria broth (LB) medium supplemented with 0.1 mg ml-1 kanamycin and 0.15 mg ml-1 rifampicin and shaken to early log phase (about 36 h). The culture was centrifuged at 12000 × g in a microcentrifuge for 10 min and the resultant pellet resuspended in 100 µl cell suspension solution (see appendix 1 B). Added to this solution was 20 µl of a 20 mg ml-1 lysozyme. This was mixed gently and incubated at 37 °C for 15 min. Then 200 µl of cell lysis solution was added and mixed completely by repeated gentle inversion of the tube. The phenol solution (50 µl) equilibrated with 2 volumes of cell lysis solution was added and the mixture was vortexed. The neutralization solution (200 µl) was then added and the contents were mixed by repeated inversion of the tube. The mixture was then centrifuged at 12000 × g for 5 min, the acquired aqueous phase was transferred to the second eppendorf tube and to this was added 2.5 volumes of 95% ethanol and contents place on ice for 10 min. The DNA was spun down by centrifuging at 12000 × g for 5 min. The pellet was resuspended in 50 µl TE buffer and stored at -20 °C for further use.
We always use the regular DNA extraction kits like PureLink DNA extraction kit from Invitrogen to extract plasmid from Agrobacterium in our lab and then back transform into E.coli for amplification and other downstream processes required. And it has always worked perfectly well.
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