The pSoup vector is a low copy vector in both E. coli and Agrobacterium -- so low plasmid yields are to be expected. I have also experienced some challenges with the Tetracycline resistance conferred by the pSoup vector.  It can be a little tricky, -- if the tetracycline level is to high -- you can't recover any colonies (and obviously if it is to low, everything grows). Recovering Tet resistant Agrobacterium with transformation of the pSoup vector alone can be done -- but you may need to find just the right level of antibiotic, in our experience that is usually about 5 ug/ml of Tetracycline (but this can depend on the Agrobacterium strain you use and media as suggested by David Oppenheimer -- concerning the Mg2+ concentration).  

Usually, my lab just performs co-transformation with the pSoup and pGreen vectors in one shot and then selects using only 50ug/ml kanamycin selection (we do not select with Tetracycline). This usually works just fine because the kanamycin selection is robust and pGreen cannot replicate in Agrobacterium without pSoup, (so recovering kanamycin resistant colonies is sufficient to select for co-transformation of both plasmids).  We use either heat shock or electroporation, and both can work fine (although electroporation typically will produce many more colonies due to higher efficiency).

Note, although tri-parental mating can be used for other binary vectors, I do not believe it works with the pGreen vector, because that vector lacks the necessary "tra" sequences for a successful mating transfer. 

Hi. Although I have no direct experience with the two vectors you named, I have extensive experience with other Agrobacterium mediated plant transformation vectors. First, many binary vectors that can grow in both E. coli and in Agrobacterium have moderate to low copy number. This appears to increase stability of the vectors in Agrobacterium. Thus you should not expect to get high plasmid yields when growing these vectors in E. coli. Second, many of the growth media for Agrobacterium contains Mg2+, which can mess up the tetracycline resistance if you are using it to select transformed Agrobacterium. For tetracycline selection, we use 10 µg/ml for E. coli, and 2.5 µg/ml for Agrobacterium. Remember to alter the Agrobacterium growth medium to lower the Mg2+ concentration to 1 mM or less (some people eliminate it altogether when doing tetracycline selection). We use electroporation for co-transformation, and use about 1 µg of each plasmid. This is overkill, I know. 

Have you tried electroporation or tri-parental mating to co-transform the pGreen and pSoup. I think that will help.

Hi David, I have worked with other agro strains and vectors and also thought the low yields could be due to low copy number plasmids. I just thought it was strange because the pGREEN concentrations were good. Thanks for the tip about the Mg2+ I am definitely going to check that out. I am using similar Tet concentrations, thanks!

Thanks Kamal, I have done both electroporation and freeze-thaw method. I have not tried tri-parental mating and will look into it.

Hi, I was using YEP media for culturing EHA105 and tetracycline concentration was 10 µg/ml for both E. coli and Agrobacterium transformation. pGREEN and pSOUP was easily co-transformed in both the strains. One thing you can do is Check the quality of your competent cells and always use fresh plasmid for transformation. 

It should not be difficult, you can increase the plasmid concentration or else do sequential transformation.

Hello, First, I transform Agrobacterium (C58 strain) with 10 µg of pSoup (10 µl of pSoup (max 10 µg) to  150 µl agrobacterium). The selection antibiotics are : Tetracycline 10 µg/ml, Gentamycine 25 µg/ml, Rifampicine 50 µg/ml.

Then, I transform Agrobacterium with 2 to 10 µg of pgreen plasmid ( 15µl max of plasmid with 30 µl Agrobacterium).

The pSoup vector is a low copy vector in both E. coli and Agrobacterium -- so low plasmid yields are to be expected. I have also experienced some challenges with the Tetracycline resistance conferred by the pSoup vector.  It can be a little tricky, -- if the tetracycline level is to high -- you can't recover any colonies (and obviously if it is to low, everything grows). Recovering Tet resistant Agrobacterium with transformation of the pSoup vector alone can be done -- but you may need to find just the right level of antibiotic, in our experience that is usually about 5 ug/ml of Tetracycline (but this can depend on the Agrobacterium strain you use and media as suggested by David Oppenheimer -- concerning the Mg2+ concentration).  

Usually, my lab just performs co-transformation with the pSoup and pGreen vectors in one shot and then selects using only 50ug/ml kanamycin selection (we do not select with Tetracycline). This usually works just fine because the kanamycin selection is robust and pGreen cannot replicate in Agrobacterium without pSoup, (so recovering kanamycin resistant colonies is sufficient to select for co-transformation of both plasmids).  We use either heat shock or electroporation, and both can work fine (although electroporation typically will produce many more colonies due to higher efficiency).

Note, although tri-parental mating can be used for other binary vectors, I do not believe it works with the pGreen vector, because that vector lacks the necessary "tra" sequences for a successful mating transfer. 

Roger may be true with this.

I agree with Roger. For completeness, this is what we do:

I use pGreen derived vectors routinely for transient transformation (Dual Luciferase assays) and Agrobacterium GV3101 (but this should also be fine for EHA105). Grow pSoup in Ecoli Tet10-  make sure the Ecoli strain you are using doesn't have chromosomal resistance to tet. Some strains do - e.g. Novablue, XL1 blue. Prepare a plasmid miniprep - kits are good, so the template is clean and free of salt for electroporation.

Agrobacterium - Electroporate cells with BOTH pSOUP and pGreen. Recover cells, and plate on selective plates containing Kanamycin (not tet), Rifampicin, and  the antibiotic on the virulence plasmid - for GV3101 this is Gentamycin. DO NOT select with Tetracycline. This isn't required, because you will only get colonies growing when pGreen is present and replicates, and it can only replicate if pSOUP is also transformed.

Electroporation is very efficient - and the cells are simple to make.

Good luck

I think Roger is right，I faired to make Agrobacterium with pSOUP ，Tet with 10ug/ml

Hi Nicky Creux,

I was also facing the same problem but finally achieved high concentration (upto 500-1000 ng/micro L) pSOUP plasmid which is compatible with pGReen0029. I achieved the high yield plasmid by continuously streaking on LB-Agar media with 10mg/L of Tetracyclin. Agromobilisation is quite tricky. You may try electroporation with the high concentration of both the plasmid sets (pGrren and pSoup) which should be eluted in sterile water, after transformation you can plate the cells on LB-Agar medium with 50mg/L Kanamycin and 5mg/L of Tetracyclin. Excess concentration of antibiotic in the medium may inhibit the growth of transformed cells.

All the best.

Hi guys,

could somebody provide me the pSoup? I have realized I need it for a binary system transformation...

Thank you

Hi All,

For my experiment of co-transformation I need pSoup vector. Could somebody provide me pSoup vector?

I will be very thankful.

Thanks

Ramesh