Never omit antibiotic! That makes it far more likely you had satellite colony contamination. Plasmids can get lost over time, so you should keep that selection pressure on continuously.

What antibiotic was used, and at what concentration? Any chance your miniprep kit/reagents are bad? Have you tested them recently with other plasmids?

I'm using Amp at 50µg/ml. For my Kit it work well with other plasmids..

How many clones selected for extraction? you may have selected only satellites that are false positives. I always check colonies by pcr before extraction and also use antibiotics at 100ug/mL which decreases chances of having false positives.

If you are worried about satellite colonies, switch to carbenicillin, add fresh to plates, and use at 100ug/mL. Limit plate incubation time to 16hrs at 37C.

However, I don't necessarily agree that this explanation fits your observations. If these were true satellite colonies, they wouldn't grow in liquid culture with antibiotics. Instead, you may have contamination with another antibiotic resistant microbe. Look at your colonies carefully to make sure that they resemble E. coli.

Colony PCR is a good suggestion. This will save time in picking the wrong colonies, and helping you diagnose your problem. Colony PCR is more informative than PCR on your ligation mix.

OK I'm testing always 12 clones. By chance I selected only false positive clones !! For liquid culture i grew my DH5alpha without antibiotic in 1ml LB for 1-2 Hours.

Never omit antibiotic! That makes it far more likely you had satellite colony contamination. Plasmids can get lost over time, so you should keep that selection pressure on continuously.

Totally agree with Daniel, never omit the selctive antibotic. I've used the pGEM system for many years and it's very good. I've had the same problem one or twice but you should be OK if you use freshly made plates and don't over-incubate. PCR colony screening is also good idea.

I don't think that the ligation PCR will tell you much. I found that there are better ligases than the one that comes with this kit. I used pGEM using the Agilent ligation kit and transformed into NEB DH5-alpha for the best results for using pGEM.

You can perform a new transformation with cells plated on medium with slightly lower conc of Ampicilin usually 100ug/ml works well. Before going to plasmid isolation check you potential positive colonies with colony PCR. If things go alright then there is a possibility that your miniprep reagents are not working well check it with any other plasmid.

Make sure your ampiciline is fresh and it is working.

You said earlier that you grow your colonies for 1-2 hours in 1 ml LB. In my experience this is unlikely to generate detectable plasmid, at least by electrophoresis following diagnostic digestion. Typically, colonies need to be grown overnight to generate sufficient plasmid for downstream analysis. In my view, given the available info, this probably explains why you're not seeing plasmid.

I mean after transformation reaction (100 µl of Cells) I grew them in 1ml for 2 hours, then I plated this suspension on LB with amp/IPTG/xgal.

Just to clarify the earlier comment- didn't realize that you were referring to liquid culture immediately following the transformation step. You should have your cells recover in SOC medium (not LB) for 1 hour WITHOUT antibiotic. Once you select on the LB plate + antibiotic, continue use of antibiotic in any subsequent cultures.

Your troubleshooting should focus on whether you are having problems with satellite colonies or contamination by an Amp-resistant microbe (not necessarily DH5a). An appropriate negative control to check for the latter is to run a transformation with no plasmid added. If you're still seeing colonies, then you have contamination. If these negative control plates are clean, then focus on eliminating satellite colonies (confirm positive clones by colony PCR, make sure you use fresh antibiotic, etc).

Hello. I have the same problem with the pGEM-T vecotr. No plasmids or very low yield of plasmids.

First to say, I have ten years experience of molecular cloning, I have made hundreds of different clones, whatever kinds you can imagine, but I have never met the problem before. Since I started to use the pGEM T-easy, every colony starts to against me. I put a lot of energy for trouble shooting.  Now I have pin down the problem to either my inserts have special secondary structure or there are problem in the T-vector itself. There are two replication origin in the T-easy which made me very uncomfortable. I read, two replication ori can result tandom plasmids. My understanding to that is when the plasmids replicate, the two copies can not separate, they either stopped or ligated as two copies tandem, next round, 4 copies tandem. There are always a super large, genome like band in my DNA gel, but very low plasmids yield. Kill me.

@Wenbo- I don't think there are two bacterial origins of replication. One is for replication of the plasmid in bacteria, another is for production of F1 phage. These should not be interfering with each other. Have you tried a positive control insert?

Dear Slimane, 

I agree with Daniel, it seems that you're not performing liquid culture after selection of white colonies. From only one colony, just making mini prep from it, you should obtain some plasmid molecules, but not visible within gel. If you don't have your colonies but still have your mini preps, try to transform again with all your prep, then you will obtain more white colonies. 

But it will be easy to try to clone again, and select the white colonies for liquid culture and then do mini prep when achieving saturating culture! (over night!) 

I had been working too many times with pGEM-T in both versions, easy and not-easy, and it works really nice. But is a bit expensive ... indeed, I had made it by myself from a blue colony mini prep, EcoRV digestion and T-tailing!