I have a plasmid pGpd-GFP arround 7 kb. I successfully inserted my inserts (0.6 kb) into to plasmid by phusion pcr followed by cloning with E. coli. Now I would like to transform my agrobacterium strain with extracted plasmid DNA (binary vector system). But I was not successful by using freeze/thaw shock transformation (by using liquid nitrogen) as described in protocol below. Is their any modified way I can do that except electroporation?
My future plan is to transform fungi with this transformed agrobacteria strain.
http://www.indiana.edu/~pikweb/PDFs%20and%20protocol%20files%20/agrotransform.html .
Transformation with purified plasmid can be done with either electroporation or a simple freeze/thaw transformation method. Alternatively, a mobilizable plasmid can be placed into Agrobacterium using the triparental mating method.
1. Did you use the right kind of antibiotics for selection (after transforming the Agrobacterium)? Have you double-checked it? What bacterial selection gene on the plasmid?
Hello Abu Bakar Siddique ,
electroporation is more efficient to transform bacterias. I use electroporation to transform Agrobacterium rhizogenes and it works for my 13kb plamid. However, making competent Agrobacteirum cells is easy but quite long. Your protocol seems okay.
Transformation with purified plasmid can be done with either electroporation or a simple freeze/thaw transformation method. Alternatively, a mobilizable plasmid can be placed into Agrobacterium using the triparental mating method.
Hello,
perhaps you could try to prepare more competent Agrobacteria by a simple (centrifugation) enrichment procedure. Unfortunately, I have no original literature about this protocol, but this is shortly what we did (successfully) with A. rhizogenes 15834:
- make a 10 ml overnight culture in LB or CPY (works better for our strain)
-distribute the sample to 5 x 2,0 ml Eppendorf-Tubes
-zentrifuge 5 min at 3000 rpm at 4oC
-re-suspend the pellet in 2 ml 10 % glycerol (ice-cold)
-zentrifuge 5 min at 3000 rpm at 4oC
-re-suspend the pellet in 1 ml 10 % glycerol (ice-cold)
-zentrifuge 5 min at 3000 rpm at 4oC
-re-suspend the pellet in 200 µl 10 % glycerol (ice-cold)
- make 50 µl aliqouts and freeze them in liquid nitrogen
- store the bacteria in -80 oC till usage (transformation)
Sometimes using the freeze thaw method the plasmid does not express antibiotic resistance in the new host (Agrobacterium) for several hours. You might try growing the transformed cells overnight before plating them on selective media.
I can send a protocol for electroporation. Do you have access to electroporation?
Electroporation of electrocompetent Agrobacterial cells would be the method of choice and is invariably successful. Plasmid preparation and cells need to be free from salt to avoid arcing, and as suggested by Yuan-Yeu Yau, use appropriate antibiotic selection, since plasmids are maintained as mostly single/low copy in Agrobacterium cells, it may help to slightly reduce the antibiotic concentration and incubate for longer periods 24-36 hours for colonies to appear. Good luck.
You should first check if the plasmid extraction have worked and what are the concentration of your extracted plasmid DNA. Secondly, how fresh are your competent Agrobacterium cells? Once you verify these two steps that you have 50-200 ng/ul of plasmid DNA and your competent cells are not more than six months old. You should try freeze thaw method and it should work. Otherwise, electroporation is the most efficient of transforming bacterial cells. If you want an optimized electro-transformation protocol for Agrobacterium, feel free to ask.
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