I suppose you transformed a binary vector into Agrobacterium. Most binary vectors are large and have low copy number in E. coli, and the plasmid yield extracted from Agrobacterium is usually even lower and very dirty. I suggest you take some of the plasmid extracted from Agrobacterium, and use it to re-transform E. coli, and then extract the plasmid. Then, do all this restriction analysis. No point doing restriction analysis on plasmid extracted from Agrobacterium because of the low yield. And, yes check the Agrobacterium transformants by colony PCR first.

Theoretically the resistance gene could be on chromosomal DNA or your plasmid extraction was not efficient. Or your kanamycin did not work, as Sezer Okay suggested.

The introduction of the kanamycin cassette into the genome of the organism results in permanent resistance to kanamycin. However, please note that the introduction of the cassette other components of the organism's genome are also affected. By coincidence or perhaps by proximity, alteration of other antibiotic targets can take place.

thanks,

I want to check antibiotic and plasmid extraction efficiency

but Mr Okay, they aren't satellite colonies because they have grown in LB broth with same antibiotic concentration.

there are two possibilities that first you check for kanamycin that it working properly or not and if it is working properly so second thing is that procedure or reagent you are using for plasmid dna isolation is correct or not and it is also possible that in agrobacterium you are transfering is resistance for kanamcin...

so check for that and i hope you may find your answer...

and resistance property is due to chromosomal and plasmid DNA both so there is possibility that agrobacterium have kanamycin resistance gene in its chromosomal gene... and you can check for it by growing agrobacterium on freshly prepared kanamycin plate....

A over incubated plate gives satellite colonies but you could easily tell by their size. Either your kanamycin is bad or plasmid isolation is not efficient. Make some fresh plates and try. Streak your Ag culture to pick single colonies. Good luck

Because the kanamycin cassette may have been introduced into the bugs genome, and it is a cassette, many culturing conditions may cause the "pop out" of the cassette, as is the case for many cassette carrying antibiotics resistance genes (example MecA for S aureus). Microenvironments may exist in your plate if the plate was not homogeneously made. Hence, some colonies may show evidence of kana resistance and others may not due to the popping out of the cassette.

I suppose you transformed a binary vector into Agrobacterium. Most binary vectors are large and have low copy number in E. coli, and the plasmid yield extracted from Agrobacterium is usually even lower and very dirty. I suggest you take some of the plasmid extracted from Agrobacterium, and use it to re-transform E. coli, and then extract the plasmid. Then, do all this restriction analysis. No point doing restriction analysis on plasmid extracted from Agrobacterium because of the low yield. And, yes check the Agrobacterium transformants by colony PCR first.

I agree with Hann's advice. This is most likely a problem with your plasmid isolation. Common issue with low copy number plasmids, and with certain types of prokaryotic hosts.

Hi,

even after changed antibiotic, the colonies Grew well. so the antibiotic checked and it was not problem.

I think Hann's suggestion is interesting and I want to try it,

thank you

true, we also always re-transform the plasmid into E.coli

Hi,

after re-transform E.coli with extracted plasmid from agrobacterium,Surprisingly we saw the trasformed E.coli grow on kanamycin(50ug/ml) but after plasmid extraction there is no plasmid.!! what's the resistance reason?

I can only speculate the problem is the low yield of the low copy plasmid. A 2-ml culture of E. coli can easily give 20ug of pUC18, but a 10-ml culture only give about 0.5 ug of a large binary vector (20kb). If you dissolve the large plasmid in 50ul of TE, then the concentration is about 10ng/ul. If you start with a 2 ml culture, this become about 2ng/ul. You need at least 10-20 ng for a proper detection of the plasmid band on an EtBr-stained gel. So, start with a larger culture, dissolve in lower volume of TE to get higher concentration, and quantify your plasmid first, before running your gel.

There are only 3 possibilities I can see:

1) As Hann said, mabye you simply are working with a low copy #/low abundance plasmid. Check your DNA prep by PCR to see if any of the expected sequence was recovered, even if in low amounts.

2) your plasmid harbors a toxic gene or unstable DNA sequence that is lost with time. Screen your E. coli colonies by PCR to confirm that you can detect the plasmid from the plate-based colony isolates.

3) your plasmid is integrating into the host genome for some unknown reason. Try to see if using the Stbl3 or Stbl4 E. coli strains work better (at a minimum, use a RecA deficient strain).

As a control experiment, you should check whether you can recover a control binary plasmid (empty vector) from an E. coli host. This will tell you whether the problem is related to the specific sequences you have inserted into your plasmid.

Mohammad,

Here are some suggestions

Try plating a control LBA4404 on Kan + plates with out any transformation.

a) If you see any colonies, then your stock of LBA4404 already could contain a Kan resistance gene

b) If don't see any colonies, your plasmid could be low copy number, so your yield could be low. In such a case, follow instructions for low copy plasmid purification such as larger culture volume etc.

Good Luck,

Balajee