What bateria strain are you using? Is it made by yourselves or bought commercially?

NEB 10-beta E. coli (NEB) and Top 10 (invitrogen) was used with the same result.

Your bacterial stock could be contaminated with lysogenic phages. Once you grow the transformants in liquid culture for plasmid extraction, many cels are lysed by lysogenic phage activation and you recover less plasmid. You can avoid it by transform the plasmid again in a new fresh host bacteria free of phages.

That is very common. The culture from fresh and newly transformed colony often gives higher plasmid DNA yield that that from old colony or glycerol stock. It could be related to the copy number, but I do not know the reasons. Doing new transformation will improve the yield.

Thanks for your response. We transferred several times into the new bought bacteria, with the same result. New transforming is a solution, but you know in every transformation there is a chance of mutations and we have observed that for every transformation all the clones behave not the same in our experiment.

There can be several reasons or all together: 1) 16kb is a huge lenght for the plasmid. Such huge plasmids are usually unstable because of recombination process in bacteria. 2). In non-persistent (as storage at -80, freeze and thaw) conditions bacteria use to integrate gene of antibiotic resistance into their genome and then throw out the plasmid. So even in presence of antibiotic they still keep growing well but without plasmid.

Sometimes they do it even if they are freshly transformated and there is the case that something in the plasmid they dont like. For exmpl the protein you are going to express. Then selection goes against weak bacteria with target protein and for bacteria with altered protein (or altered plasmid) or for bacteria which integrated resistance gene in the genome.

To control the expression in bacteria,for example you can use special strains which already express supressors for target plasmid promoter (for example JM109 contain "inhibitor" for lac-O) and supress expression when growing the bacteria for maxi (for example if you got promoter under lac-O the addition of glucose will strongly supress the expression). This will stabilize the plasmid in bacteria.

You dont need to bye new bacteria every time. You can make glycerol stock of plasmid free bacteria and store it -80 with apropriate antibiotic if needed. And make them freshly competent any time you want.

Good luck!!!

The issue might be a very simple one, since the bacteria are not dividing very actively during the glycerol storage. also, during the freeze thaw, we might be losing on some bacterial cells as well (maybe around 5% or so). Additionally, if your plasmid holding capacity requires to be tightly induced, is your glycerol stock medium containing that specific dissolved amount of the inducer?

Hope this might help you ahead....

The answer may be a very simple one. Did you use just a loopfull of the glyc. stk. or 0.5-1 ml of it in your culture? The loopfull would be best -- a high glycerol % in a miniprep culture can sometimes hurt you. Good Luck, Ron.

16kb plasmid is enormous and prone to mutate in regular competent cells such as NEB 10-beta or Top10. Especially when you are dealing with lentivirus constructs because of their long repeats, which can be considered as damaged or foreign by bacteria DNA repair system, and therefore "repaired" (altered). The SURE strain from Agilent or Stbl2/3 from Invitrogen have been genetically altered to remove some their DNA repair system, thus result in much less mutation rate for unstable constructs. Stbl3 also grows at 30°C to slow down growth, which further stabilize the plasmids.

You may consider a better way to freeze your bacteria http://www.tantecbiosystems.com/news/.

Glycerol media is not the best and has a tendency to cause plasmid loss. Use a richer media as outlined in " the cloning manual" to grow your plasmid, Your plasmid is also quite big and you may consider a alternative bacteria background that can stably handle even larger plasmids as those used to BAC cloning.

Good Luck with your research

We have similar problems. My old PI once explained why but I can't remember exactly now. Something about the plasmid being so large that you need the most robust bacteria and glycerol freezing reduces activity/protein production such that by the time the bacteria are "up to speed" the plasmid hasn't even divided yet. I understand your concern about random mutations and every clone yielding slightly different results, but you will find this to decrease if you switch to a bacteria with reduced recombination abilities, as Kejun suggested. We use Stb3 to grow our lentiviruses, transform fresh each time, use a fresh colony from the plate, do not use a "starter" culture (excessive culturing increases the likelihood of spitting out or recombining the plasmid) instead culture the single colony from the plate straight into your maxi sized broth. Use a very rich broth like 2X YT, use double the antibiotic you normally use (we use 100 ug/ml amp), culture overnight (between 18-22 hrs, no longer as this may allow bacteria to kick out plasmid while recombining amp resistance), slow rpm (150-200, again the plasmid is so large, it needs time to replicate). All these conditions will ensure your clone does not mutate but the yield will always be lower than what you get from smaller, pcDNA-type plasmids. Keep in mind these are huge plasmids. I use this protocol with 100 ml 2X YT and typically get OD readings of only 1.5-1.8. After prep (I use Nucleobond Midi Extra with Finalizer with best results) and get 500-1500 mg total (which is expectedly low). After RE digests to check (always, always), my clones are almost always perfect. I also often sequence clones that have increased chances for mutation (with shRNA, etc). Oh, and I never do less than 2 midis/clones at a time, in fact, I often choose 5-6. Many of my lentis contain genes for color and despite the fact that bacteria are not supposed to be able to make these proteins (based on promoters), the bacterial pellets of good yields will be green or red. I say this so you will take extra safety precautions during processing - if they can make GFP, they may be able to make other lenti/HIV proteins.

Thanks Emily, I will try it .