Low yield of plasmid DNA

[lina W]

Hello,we have constructed several TALEN in pTALEN backbone using Zhang lab protocol. We have problem to get high yield of plasmid DNA that we would need large amount od DNA for several transfection in eukaryotic cell. We get maximal 3µg of plasmid DNA by inoculating 5 ml of LB for 24hr at 30°C.

Is it a low copy plasmid? 

Thanks for your advise.

lina W

[Feng Zhang]

Hi, what kind of competent cells are you using? We usually use Stbl3 and have good yield.

lina W

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[lina W]

Hi, we are also using Stbl3. We incubate them at 30°C for 24h instead of 37°C to reduce the risk of mutation. We get a large bacterial pellet but low DNA yield....

Lina W 

[natha...@gmail.com]

Hello, I know it was ages ago but I am having this issue currently and wanted to know if you ended up figuring it out.

Thanks

[Le Cong]

I think you may want to switch to use 37C for overnight / 12-16 hours, because we haven't encounter series issue with recombination when using stbl3 at 37C (though it can happens occasionally). 

One issue with long incubation is loss of selection pressure over time if you use ampicillin (which is what you probably used if you use the Zhang lab TALE system?), then the plasmids will be lost resulting in low yield despite sizable bacterial mass.

Best,

Le

Le CONG, Ph.D. 丛乐

Broad Institute of MIT and Harvard,

Massachusetts Institute of Technology,

415 Main Street, Cambridge, MA 02142

Tel: (+1) 617-714-7879

Mobile: (+1) 617-823-6132
E-Mail: congl...@gmail.com / co...@broadinstitute.org / con...@mit.edu

On Wed, Jul 29, 2015 at 2:52 PM, <natha...@gmail.com> wrote:

Hello, I know it was ages ago but I am having this issue currently and wanted to know if you ended up figuring it out.

Thanks

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[natha...@gmail.com]

Sorry I forgot about that detail. I did grow them up at 37˚C overnight but my range has still only been 1-3µg for a 7ml culture. I think I will just suck it up an do a large midiprep at 25ml+. And yes I am using the Zhang lab TALE system with an amp.

Thank you,