Hi Zeesham,

The rearrangement of transgene cassette on binary vector may cause problem too.

1. The rearrangement (such as deletion) might have occurred and deleted your gene cassette in Agrobacterium. For example, if you have homologous DNA sequences or repeats (especially the long repeats) which flank your gene-of-interest (GOI) (for example). After this binary vector was transferred into Agrobacterium, there is a chance that this GOI be looped out through homologous recombination. If this is a case, then: (1) when you do PCR (assuming you are PCRing GOI), the 'original' cloned plasmid will give you a band, but the mini-prep DNA from Agrobacterium will not generate a band.  (2) Even your GOI is deleted, the Agrobacterium still harbor the 'leftover' binary vector backbones and the backbones can still replicate In the Agrobacteria (with kan resistant). Therefore, the Agrobacteria with these leftover backbone can still happily grow on medium with Rif + Gent and Kan. 

2. That is the reason why instruction sheets from some companies producing competent cells (especially E. Coli) will tell us the genotypes of the particular bacteria they have used in the kit. One of the important genotypes is 'recA', a mutated RecA. With recA, the risk of our plasmids been modified (rearrangement through recombination) in those cells will be reduced. 

3.Except this RecA gene, there are other mechanisms involved in rearrangement of  T-DNA. Try to avoid using too many long homologous DNA in the construct.  I attached a paper for your reference.

Good luck on your research,

Sounds like your PCR is not working. Are you trying to amplify the resistance gene? Do you get measurable amounts of DNA from the minipreps? Maybe a restriction digest of the minipreps can help you, although I find it hard to imagine that you do not have transformed agros when they can grow on triple selection! How did you transform you cells?

We used Heat shock method for transformation. . And in PCR we used a positive control of the same plasmid (containing our gene of interest) extracted from E.coli which gave positive results.

It does sound weird indeed! I assume that your plasmid is the Kan selection? Try to plate your cells on Rif gent and kan plates individually. That way you can evaluate if it is only your plasmid that behaves oddly or its also the Transfer DNA plasmid. Alternatively, try electroporation and see if you get the same.

Strange it is really! Positive is working, colonies are growing well on antibiotics...

Why you are going for prep here? Why not colony PCR or broth PCR??

Two things further you can do by the way, change the method of transformation of agro cells. Go for electroporation, rather than heat shock.

How much plasmid you are harvesting, check it quality and quantity, make further dilutions of it. It must work.

@Tonni Andersen yes mt plasmid is the Kan selection and i have already cultured cells on Rif+Gent and Kan plates individually.. The cells were on songs on both plates unlike the non-transformed control which wasn't able to grow on Kan.. @Allah Bakhsh i have tried colony PCR as well but no results :( ...

Dear Zeesham Nasim,

For Agrobacterium use electroporation method than heat shock. In heat shock u will get very less no of colonies and among them few will be positive. One more thing agrobacterium colony PCR does not work as well in comparison to E.coli. Take only frsh grown cells for PCR, very minute quantity as minimum as you can take. You will get desired bands.

Hello Zeesham,

Another thing you can do is, after isolating your mini-prep DNA, transform E.coli with it and check by the PCR or restriction digestion. In my experience, with Agrobacterium, the mini-prep DNA is neither of good quality, nor worth doing much molecular biology on it.

If you have plasmid in there, it will be transformed in E.coli and colony PCRs work well there.

All the best.

Simran

Hi Zeesham,

The rearrangement of transgene cassette on binary vector may cause problem too.

1. The rearrangement (such as deletion) might have occurred and deleted your gene cassette in Agrobacterium. For example, if you have homologous DNA sequences or repeats (especially the long repeats) which flank your gene-of-interest (GOI) (for example). After this binary vector was transferred into Agrobacterium, there is a chance that this GOI be looped out through homologous recombination. If this is a case, then: (1) when you do PCR (assuming you are PCRing GOI), the 'original' cloned plasmid will give you a band, but the mini-prep DNA from Agrobacterium will not generate a band.  (2) Even your GOI is deleted, the Agrobacterium still harbor the 'leftover' binary vector backbones and the backbones can still replicate In the Agrobacteria (with kan resistant). Therefore, the Agrobacteria with these leftover backbone can still happily grow on medium with Rif + Gent and Kan. 

2. That is the reason why instruction sheets from some companies producing competent cells (especially E. Coli) will tell us the genotypes of the particular bacteria they have used in the kit. One of the important genotypes is 'recA', a mutated RecA. With recA, the risk of our plasmids been modified (rearrangement through recombination) in those cells will be reduced. 

3.Except this RecA gene, there are other mechanisms involved in rearrangement of  T-DNA. Try to avoid using too many long homologous DNA in the construct.  I attached a paper for your reference.

Good luck on your research,

Hi Zeeshan,

Colony PCR does not work well with Agrobacterium b/c the vast majority of ori on binary vectors that function in Agrobacterium will maintain the plasmid at very low copy numbers (1-5 per cell) as opposed the ori that function in E.coli (>300 copies per cell), so the target is rare relative to cellular debris and other PCR inhibitors.  One trick you could try is to grow a 1ml overnight culture of your Agrobacterium, then spin that culture down, and then resuspend it in 100 uL of water.  You can then try 1 - 5 ul of the resuspension in a PCR reaction in which I would use a robust polymerase, not standard Taq, although if that's all you have then you'll just have to make do.  If this does not work, I agree with Simran above - miniprep DNA from the Agro will be of low yield and reduced quality, so it would probably be best to use it to transform E.coli and do your molecular analysis starting there.

Your colonies might just be yeast. We have a big problem with this due to the fact that Agrobacterium cultures have to grow for long times. How does the Agro colonies look? Colony PCR is difficult with Agrobacterium. Spin down 1 mL of your overnight culture and resuspend it in ddH20. Then boil it for 5 min and use 5uL of that in your PCR reaction. Works every time :-)

I am wondering if any updates there. I tried the liquid culture too, but the PCR still did not work with the primers either exclusively designed on vectors, or one on the gene of interest, the other on vector. The positive control using the final plasmids always worked! No idea what are grown on the plate... Any suggestions? 

Hi

I had the same problem with this strain and LBA4404. whatever is the reason(deletion, recombination,...) it is better do not waste your time for miniprep... just grow single colony over night in 1-3ml YEP broth+selection antibiotic till reaches O.D600~1.

Take 100ul and wash 2-3times with SDW ,

then add500ul SDW and resuspend  throughly.

Boil 10min and then centrifuge for 30second/13000 rpm to pellet the cell debris.

take 5ul supernatant for 20-25 ul PCR reaction. if PCR result was negative then be sure the grown Agrobacterium  on both solid and in broth even containing selection antibiotic is negative.

Good Luck

Thanks dears  all for your comments on this question.

Indeed, I faced same problem now. But, I got positive colonies by using high fidelity enzyme rather than normal enzyme for pcr. As the extracted plasmid Con.from agrobacterium very low, therefore high fidelity enzyme works well. I think no need to boil the bacteria, it doesn't work with me.

@ Nikhilesh Dhar,

1. Regarding your question: By the way what is the genetic background of GV3103?

Please see the attached 1986 paper, Table 1. It also listed an relevant paper about GV3103; you can check on it.

2. But judge from Zeeshan's selection medium set up (Rif+Gent+Kan), I think that he was using GV3101 (as indicated in the question title). GV3103 in the question subtitle could be a typo.