How did you do transformation ? It is hard to know  why, better to re-transform again and check it. 

in this case i suggest you to run colony PCR, use specific primers from your vector (eg. T7 pro and T7 ter primers for Pet 28a). then you will know whether the problem is Plasmid isolation or no transformation. at the same time use cell free plasmid as control.

So many things to be asked here?

1. Did you do transformation before extracting the plasmid?

2. If you did the transformation, is it successful? How about positive and negative control?

3. Let say your transformation is a success. did you already confirm the insert gene inside the plasmid either by double digesting and colony PCR?

Do you know that the resistance phenotype depends on a plasmid? If yes, what type of plasmid did you use for cloning, or, if you are looking for a natural plasmid, what type of plasmid do you expect? How do you know about the 8 kb size? Can you make a few more remarks on the organism that you study?

I am using Top 10 strain and transformed into ampicillin culture. Trying to extract AAV  plasmid (ampR gene into plasmid). I want to make large stock for that plasmid so I have every details of plasmid. I am using alkaline lysis method follow up by phenol-chloroform purification for plasmid extraction.

Bacteria are notorious for incorporating genes.  There are papers where bacteria downstream in a flow cell had picked up genes from upstream cells.  It could be that your bacteria picked up the antibiotic resistance without the plasmid.  Or it could simply be a mutation that made it grow.  Does it grow normally?  Or is it slowed?

Bhoj and others have it right.  Best to try to transform another batch and see if you have the same problem. 

I transform bacteria, then they are grown on the solid selective media (with agar on Petri dishes), then I choose antibiotic-resistant colonies and transfer them on new Petri dishes with antibiotic.

To extract plasmid DNA, I grow bacteria in liquid media without antibiotic.

Such method is used for plasmids carrying gene for resistance to ampicillin (pay attention to the mode of resistance to this antibiotic!!!) and can be used for other plasmids.

If you study other antibiotics (e.g. tetracicline), You can add antibiotic to liquid culture, but check concentration (it can differ from concentration in solid media).

Also pay attention that Tet-resistant bacteria can be free from a plasmid or loose a plasmid; they can carry resistant gene in a chromosome.

Could you give more details about your plasmid prep?

Please tell us which method you're using to purify plasmids

Dear Mohit,

Please tell us about your bacterial strain and also the protocol u are using for plasmid extraction?

Antonio and Samaneh,

Bacterial Strain - Top10 bacterial strain

Protocol - Alkaline lysis method follow up by phenol-chloroform purification and I also used column purification. 

Hello Mohit,

If your plasmid exists at a low copy number in the cell, you may have very little quantities (not enough to be seen on a gel).

The alkaline lysis/phenol-chloroform method will probably give you lower yields than a column kit.

To improve the yield with a column kit, you may try these steps instead of the provided protocol (it is the protocol I give my students):

- The overnight culture (maximum 16 hours) should be 10 mL in LB. Use antibiotics to avoid loss of the plasmid. Carbenicillin is less rapidly degraded than ampicillin.

- Pellet the cells (10 minutes 5000g) in a tube with a conical bottom (Falcon)

- Remove supernatant, then invert the tubes on absorbant paper to remove all the culture media, then pipette the rest. No liquid should remain visible: this is really important.

- Resuspend (in resuspension buffer) carefully the cells by pipetting and vortexing a lot (it may take some time).

- After addition of the lysis buffer, invert slowly the tubes for 1 or 2 minutes. The liquid shoud be viscous.

- After addition of the neutralisation buffer, invert the tubes for 1 or 2 minutes. The solution looks like coconut powder in a transparent liquid.

- Centrifuge for 10 min at 13000rpm. The white pellet should be firm.

- Transfer (pour, then pipette the rest) the liquid to the column, and then centrifuge 5 minutes at 5000 rpm. The protocol provided in kits tells to centrifuge 1 min at 13000 rpm: however, 5 minutes at 5000 rpm works really better.

- Wash with wash buffer (centrifuge 1 min 13000 rpm). It usually contains ethanol. If you had to add the ethanol to a concentrate, make sure that you are adding absolute (100%) ethanol, and not 70% ethanol or modified 90% ethanol. Only absolute ethanol will work.

- Remove the wash buffer from the collection tube. Centrifuge again 1 min 13000 rpm.

- Remove what is left of the wash buffer from the collection tube. Centrifuge again 1 min 13000 rpm.

- Let the tube open for at least 1 minutes to evaporate the remaining ethanol.

- To elute, add 50 µL of ultra-pure water heated to 70°C, directly to the centre of the membrane. Wait 5 minutes at room temperature. Then centrifuge 1 min 13000 rpm.

With this protocol I usually get yields 2 or 3 times higher than with the protocol they put in the kits. I used this protocol with very cheap column kits and it worked well, even with low/medium copy plasmids.

You can also try to double the volume of the first 3 buffers (resuspension, lysis, neutralisation). The total volume will be too big for a column, so fill the column, centrifuge, fill the column again until all the sample has been passed through the column.

But the only way to make sure that you had a plasmid in the first place would be to do a colony PCR with primers specific for your plasmid. You just have to resuspend one small colony in the PCR mix, the initial denaturation step at 94° or 98°C for 2 to 5 minutes will lyse the cells.

Dear Mohit, you might check again your antibiotic and your bacterial strain. An unscucessfull selection could lead to the growth of mutants or also the cells could have contaminants resistant to the antibiotic. If you confirm with PCR and even you can´t extract DNA, you should try a better purification method. The previous protocol should be correct, it´s very important to use 100% etanol on the wash buffer. If it has been already prepared and you aren´t sure, you can also use only absolute (100%) ethanol as wash buffer. Also, you can elute the DNA with warm water (up to 60ºC).

If you still have problems after using this protocol, you can extract DNA from a 50mL bacterial culture. Let me know if you want me to send you this protocol.

Good luck

1. PCR confirm the bacteria do have the gene on the desired plasmids

2. If you still have the plasmids, try to make new competent cells and transform again

    do a control experiment to make sure your competent cells are not resistant to the selection markers that you use

3. confirm the version of the replication origin of your plasmid

    You should know whether you are using a low copy number or high copy number

    plasmid

4. test your antibiotics and make a new one

I have 2 related questions. Can I plated bacteria on solid media plus antibiotics and then use these bacteria for extraction their plasmids? In the liquid media most of my bacteria dont grow up even without the use of antibiotics.

In stage 1, in most cases I have to vortex the samples a long time but the colonies still are visible and they dont get unfolculated. what should I do?

Thanks for your advice. Yes, they grow on solid medium not in liquid. Maybe that s because they don't be shaken in incubator, I m not sure.

I meant in stage 1 mentioned in the kit we should have a white suspension and no colony should be seen but I see colonies.

I did t transform plasmids. I just plated bacteria on a plate with a high concentration (0.030 μg/ml) of 4 antibiotics and then isolated grown bacteria. I wanna see if their resistance genes are located on plasmid or chromosome. So, first of all,  I wanted to see if they had plasmids. The bacteria are unknown but they are probably from Pseudomonas spp. (because of use of PSI medium).

Really sorry. 0.030 g/ml or 30 μg/ml.

Yes I do. The medium was cool (40 c) and the used antibiotic was tetracycline. I plated them on liquid medium today, again. I m waiting for the results.  Really thanks for your answer.

I plated them from soil on solid medium plus antibiotics and then isolated them on solid medium without antibiotics and the inoculated them on TSB plus tetracycline. But still have t detected any plasmids.

really thanks. I ll try it.