It is possible but not likely that your insert will be affecting the copy number, although I doubt it would be 10-15 fold reduction. Is your insert expressing a protein in E. coli that might be somewhat inhibitory to bacterial growth?

Do the mini preps work well with this insert, you only mention maxi preps?

Dear Antonio

in which e.coli strain did you perform the cloning? and the plasmid extraction? which is the promoter of your plasmid?

did you see differences in terms of bacterial growth and biomass? e.g the amount of e.coli cells that you obtain for plasmid extraction with the plasmid with insert is lower than with out insert.

because i think that he insert can affect the yields if:

1) you are using an e.coli strain where the insert can be expressed and if the protein expressed it toxic to the cells.

in this case the cells are not able to growth well and therefore you extract less plasmid just because you have less cells and not lower plasmid on each cell.

2) the protein that you expressed has a function that affect plasmid replication or stability... (eg is a beta-lactamase or dnase ) and in this case can affect the plasmid stability during replication and therefore the final copy number.

one other possibility is that the efficiency of your kit is not so good for big plasmids.

In my experience the yield decrease with plasmids >10kb.. your size is still acceptable but you can try to use a different kit.

in this context i would like to suggest you the Omega biotek mini and midi kit (you can buy it from VWR) that in my experience supply amazing yields.

you can find some data about it on my blog: https://proteocool.blogspot.com/

proteocool n°12

other possibility is use a kit based on magnetic beads and not coloumn that in case of very big plasmid can be less efficient because plasmid is top big to pass into the resin.

good luck.

ciao

Manuele

I agree with Michael that expression of a toxic protein may be the problem, since it will favor the selection of low-expressing low copy number segregants. I don't know what sort of construct nor what kind of purpose you plan to use it for, so it is difficult to suggest further advice. However, if you plan to produce a toxic recombinant protein, you may try an expression system afforcing stringent control of expression, such the arabinose-inducible strain BL21AI.

In addition to toxicity/interference with replication by insert-encoded proteins, there is the possibility of interference with replication by runoff transcription from a promoter located in the insert without a matching terminator. See e.g.

Article Mechanisms of Transcription-Replication Collisions in Bacteria

But I agree with Michael's suggestion of first checking if the construct also exhibits poor yields in minipreps.

Thank you all for your answers.

First, Michael J. Benedik , I have cloned the cas9 gene downstream two lox sites, so toxicity should not be the problem because there should not be gene expression.

Second, Manuele Martinelli , The original E. coli strain of the backbone plasmid was the Dh5a strain. For the cloning I used E. coli Stellar cells. After that I made a glycerol Stab to conserve the plasmid (which i used for the two maxiprep that yielded poor DNA amount). I have already re- trasform the plasmid in Dh5a strain which should be the appropriate one, but minipreps using this colonies yielded really low DNA amount (60 ng/ul , I usually get between 400 and 700 ng/ul).

Third, I will try this week to perform a miniprep with the stellar strain that i used for the Maxipreps, to see the ratio between maxi and miniprep.

Last thing, I did not notice big differences in term of biomass in the bacteria pellets.

Would you please forgive me if my english is not perfect , I'm trying to improve it.

Minipreps confirm that my plasmid has 10 fold decrease in copy number. My positive control yielded 700 ng/ul while my plasmid was just 75 ng/ul. I probably need to sequence the oriC, searching for mutations.