Hello,

I think, when you obtain the colonies after transformation, either you should inoculate each of them (only the fresh colonies) directly into 25 ml or you should take an aliquot for secondary inoculation at O.D. 0.6. because at O.D. 0.6 bacterial cells are in the log phase.

Also, sometimes it happens that we cannot see the plasmid precipitate but it is still there (happened with one of my colleague) so maybe you should go for the last step and dissolve the plasmid in TE buffer and then go for quantification . If you have done it already then I will go with my above suggestion.

Maybe you should firstly check the colonies by manual mini-prep., then you can use kit. If plasmids are not correct or they have never been obtained, you might not have wasted midi-prep kit. By the way, I think Ethanol is more powerful precipitator for nucleic acids.

Dear Alessandro

Sometimes, the pellet after isopropanol precipitation is not huge and you can see it well only if you are centrifuging in trasparent tubes as falcoon end only once the samples become dry after isopropanol removal.

However why are you using midi kit at this stage? Is it a plasmid for mammalian cell trasfecion and you need to prepare it in high amount?

Or you have just to send it to sequencing and trasform it in a e.coli expression strain??

I can suggest to you to use the "ezna hp mini kit II" from omega biotek ( distributed by vwr) that starting from an o/n 10 ml lb culture will suppliy to you yields close to the qiagen midi kit but saving time and avoiding the isopropanol precipitation step.

From the same supplier you can found also a good midi kit that not require isopropanol precipitation but in my experience in 90% or cases their mini kit can replace a standard qiagen or invitrogen midi kit.

Moreover you can direclty inoculate the single colonies in 11 ml of lb (with antibiotic) o/n and use 1ml for prepare glicerol stock and the other 10ml for plasmid extraction avoiding the secondary iniculation.

Good luck.

Manuele

Thanks Manuele for you answer.

I'm using that kit because it is the one that gave us good result in the lab with the same plasmid (they scaled up the process cause they couldn't get enough plasmid with mini kits).

I'd also prefer to make this kit work instead of buying a new kit for now.

I ran specs of the fractions and I got absorbance at 260 for the first one (loading of pDNA on the column).

Either my pDNA doesn't bind to the column or that absorbance is caused by gDNA that I still have in the surnatant, that would impy also I have no pDNA in my cells at all even if they grown on Amp (I'd tend to exclude this cause the precipitation of gDNA in the first steps seems to be ok with viscous solution, proper mixing using Lyseblue and so on).

Assuming that simply my pDNA doesn't bind to the column the troubleshooting guide suggests that column's overloaded and to decrease the colture volume. I haven't tried this but even if the column is overloaded I should still have pDNA binded on it with any absorbance in the first fraction caused that pDNA that didn't bind to the column. I can't see any reason why, if it was the problem, I have not any pDNA at the end of the process.

SOLVED:

I was trying to purify Plasmid DNA from XL-1 Competent cells that grow much slower than normal BL-21(Gold)-DE3 normally used for expression.

The issue was that I couldn't get an appropriate concentration of plasmid DNA cause the cell I was growing where too few.

QIAGEN in fact suggests 3-4x10^9 cells/mL, from XL-1 I was getting less.

I just scaled up the growing and I could get DNA precipitate with both types of cells.