Hi Jorge,

what plasmid backbone you're using? High/medium/low copy? Try to streak your colony on fresh antibiotics plates and increase your AB-concentration in the main culture. Ampicillin doesn't kill bacteria! If you keep your plates too long the ß-lactamase from the resistant bacteria will degrade the amp in the surrounding medium (just keep a plate for 24h in the incubator and you'll see all the "satellite" colonies coming up) This allows un-transfected bacteria to grow up. If you inoculate this "mixed" colonies the non-transfected bacteria may out-grow their plasmid-carrying peers after the amp has been degraded in the culture (happens after a few hours because ß-lactamase will be released to the medium).

Cheers

Robert

Hi Jorge,

what plasmid backbone you're using? High/medium/low copy? Try to streak your colony on fresh antibiotics plates and increase your AB-concentration in the main culture. Ampicillin doesn't kill bacteria! If you keep your plates too long the ß-lactamase from the resistant bacteria will degrade the amp in the surrounding medium (just keep a plate for 24h in the incubator and you'll see all the "satellite" colonies coming up) This allows un-transfected bacteria to grow up. If you inoculate this "mixed" colonies the non-transfected bacteria may out-grow their plasmid-carrying peers after the amp has been degraded in the culture (happens after a few hours because ß-lactamase will be released to the medium).

Cheers

Robert

Hi Robert,

Thanks for the answer. The plasmid is a low copy number for Y2H. I streaked the positives colonies in fresh plates and it grows without problem. When I grow them in liquid media, LB+Amp (75mg/L), I find the same problem again.

Hi Jorge,

75mg/L amp is a bit low. We're using 160mg/L (ampicillin). We've seen that plasmids can be lost from colonies on plates within 2 days. I'd restreak a few colonies, grow them o/n and inoculate 5ml of 160mg/L LB medium in the morning. Harvest all after 8h to 10h growth (should be sufficient for a common strain like XL1-blue) and use for a miniprep. This strategy solved most of our problems.

Good luck

Robert

You should try to use mix of Amp (50ug/ml) and carbenicillin ( 50ug/l) as carbecillin is same family of antibiotic as amp but it wont be degraded and you will not see non-transformed bacterial growth. Miniprep yield is not too bad compare to your plasmid copy number but you can use more overnight culture. DO you see you plasmid without restriction analysis? sometimes you just isolate some rubbish rather than you actual plasmid. SO before doing restriction digestion , try to check your plasmid backbone in gel rather than on nanodrop.

I've had similar problems with some toxic plasmids (viral genomes) and depending one the individual plasmid at least one of the following solutions worked:

1) plasmid prep directly from agar plates (streak a retransformation on a plate, grow them, rinse with H2O and collect)

2) grow in Stbl3 E. coli

3) grow at RT

Also be careful that you don't copurify DNA-degrading enzymes like endoA with your miniprep (Use Buffer PB - or whatever it's called in your particular kit - for washing).

Hope that helps!

Best, Thomas

HI,

Try subculturing. Grow the colonies in liquid broth for 8-10 hours and then add the same liquid broth in fresh medium and culture it overnight to get a better yield. This is usually the case for low copy number plasmids.

I hope this helps.

Are you sure that your plasmid is correctly cut ? Sometimes we think yes but in fact it will close on itself.

I had the same problem in the past. And a simple solution: my ampicilin aliquots were old. Just did fresh aliquots and the problem was solved. It was growing bacteria without the plasmid.

Are you sure your mini preps are not contaminated with high salt concentrations?

How big is this plasmid?

A good thing would be to grow a control plasmid (without insert) which needs the same concentration of antibiotic for selection. If you get minprep from that and not from your clones, than you know there is something wrong with your clones.

I had this problem once. Earlier I used to do one step elution, it worked fine. But sometimes it doesn't work then you have to try two step elution. Along with that you can incubate column with elution buffer for 15-20 minutes. It also works.

Many of the previous comments about plasmid loss and making sure your antibiotics are good would be the place to start. But it may be that your problem isn't plasmid loss but rather your miniprep DNA is not good. You could be losing your plasmid DNA during some step of the protocol, or you could have nuclease contamination and it is all being degraded. Have you successfully analyzed miniprep DNA with some control or different plasmids recently?

Thanks a lot to all of you!!

My plasmid is a low copy 8Kb and my inserts are 0.2 - 0.6 -1.5 - 3 and 5Kb. None of them worked. They came from the same gene. I am doing deletions to check interactions in Y2H. I did transformation with the empty plasmid (no insert) and no problem at all. The miniprep yield was around 150ng/uL and when I digested I obtained the expected sizes. I will try to re-streak the positive colonies, grow them o/n and liquid culture with individual colonies in Amp and in Carbencillin as suggested. I am also thinking to try with Stbl3 cells but I checked and they are kinda expensive ($400 for 20 aliquots - $20 per transformation!!). Let's see if this works!

The problem could be the presence of unspecific nucleases coming from your competent cells.

When you add the restriction buffer they become more active with consequent unspecific digestion of your plasmid.

I suggest you to check before if your cells belongs to the MH series or others that are rich in nucleases.

After that, I suppose that you use a kit for your miniprep. Check if in the protocol you can do a further wash to eliminate eventual nucleases.

The fact that you can detect the desired transformants by colony PCR indicates that cloning works but that you are losing transformants during growth. Even if the transformants are lost as the bacteria in the colony multiply, you will still see PCR products of the expected size. If this is happening, even if you select the right colony (no satellite colonies) you will be inoculating your culture with a mixture of cells; some containing the desired plasmid, some with a partially deleted version of the plasmid (still Amp resistant), some cured of the plasmid surviving within the mass of the colony as the Ampicillin concentration around that colony diminishes.

As others have mentioned, Ampicillin can be exhausted during growth and once this concentration falls below a certain level, Amp-sensitive cells will grow. To check this, take two small aliquots of your overnight culture and plate one on freshly made plates with ampicillin and one on plates without ampicillin. If there is no plasmid loss then the numbers should be the same, (though even plasmids with a deleted insert will grow on Amp as long as the Amp gene is intact). You could do a simple time course, as described above, which could show you, for example, that up to 8h of growth, almost all the cells contain plasmids but after 8h an increasing number are Amp-sensitive. In that case, rather than doing a normal 16-24h growth of your transformants, you might try growing multiple transformant cultures for 8h, harvesting and combining the plasmid mini-preps.

In the past when a particular E. coli clone gives me a low yield of plasmid upon mini prep, I have found that transforming competent cells with the "empty" mini prep to get "fresh" cells carrying the plasmid almost always yields clones that carry the plasmid and give a much better yield upon mini prep.

Shortly (~30min )before your miniprep, add chloroamphenicol. This inhibits DNA but not plasmid replication and will increase your plasmid copy number per cell dramatically.

HI, you can try to grow your bacteria at 30°. I have "saved" some delicate cloning just by doing this. good luck.

What's the size of your plasmid and is it a low copy strain? If so, try extract from larger volume like 200ml and use midiprep.

Supposing you do alcaline lysis preps...just in case...Do you place miniprep on ice during incubation with buffer III? This would make SDS precipitate co-precipitating most of plasmid DNA to the pellet. Do all steps at RT! Maybe it's that simple...good luck!

This is not an uncommon problem if the insert contains a gene that causes problems for the cell. We find this often with human genes on E. coli expression vectors. The best way to do this is to use a different vector that has a Tetracycline, or Kanamycin resistance selection instead of Amp. When using Amp, we will often incubate the plates for 8 hrs, lift up the agar pipette a dilute (500 ul total vol) of fresh amp underneath to a final concentration of 100 ug/ml of agar in the plate.The amp will diffuse over time supplying fresh amp during overnight incubation.

Make a fresh stock of ampicillin and of your liquid culture medium with amp. Ampicillin has a fairly short half life and its efficacy is greatly decreased after just a few weeks of storage.

There have been a few occasions where I had problems growing a culture in liquid and instead I would pick a colony and with a toothpick make a large plate on an amp plate (or whatever antibiotic). You can smear out 6 or 8 per plate. When the smear has grown fully you can collect the cells and resuspend in solution to make a mini prep.