How much cells did you use for the extraction? The optimal amount is usually mentioned in the manual. I generally use 15OD cells for high copy and 30 OD cells for low copy plasmid. This works well, especially for tricky constructs.

Try not to overgrow your culture. If it remains in the stationary phase for too long, the plasmid will degrade. You can easily grow your cells o/n at 30°C and they will still reach confluence.

Try not to lyse your cells too long. Neutralize as soon as the liquid turns clear.

Use less elution buffer or reload your elution on the coloumn. Preheat your buffer to 60°C and incubate for 1-2 min.

These are the usual mistakes i notice in our lab. I guess the proteinase K treatment only helps with cells lysis, which usually is not an issue.

You can try the chloramphenicol method, to additionally increase your plasmid yield.

Dear Ting

which concentration and ampicilin and culture media did you use for the O/N culture? Which mini kit? Which E.coli strain did you use for propagation?

Generally with standard mini kit (eg qiagen) yielad in the range of 100ng/ul.

I may suggest to you to use the EZNA MINI KITII (Omega biotek) which contain the treatment with prroteinase K coupled with high capability binding coloukmns and in my experience supply yields about 5 times higher than the other mini kits.

(you can see some example in my blog ProteoCoool https://proteocool.blogspot.com/ at PAGE 6) but i suspect that you have some other factor that limit your yields.

ciao

Manuele

How much cells did you use for the extraction? The optimal amount is usually mentioned in the manual. I generally use 15OD cells for high copy and 30 OD cells for low copy plasmid. This works well, especially for tricky constructs.

Try not to overgrow your culture. If it remains in the stationary phase for too long, the plasmid will degrade. You can easily grow your cells o/n at 30°C and they will still reach confluence.

Try not to lyse your cells too long. Neutralize as soon as the liquid turns clear.

Use less elution buffer or reload your elution on the coloumn. Preheat your buffer to 60°C and incubate for 1-2 min.

These are the usual mistakes i notice in our lab. I guess the proteinase K treatment only helps with cells lysis, which usually is not an issue.

You can try the chloramphenicol method, to additionally increase your plasmid yield.

Thank for your answer Simon Flueckiger & Manuele Martinelli .

For the concentration of amp, I used 100 ug/ml of amp and the medium is LB, as I cultured DH5 alpha bacteria.

Therefore, in your experience, is the problem related to the overnight culture and mini-prep problem, instead of the cloning or competent cell problem?

I would like to say a big thanks to you, as this problem makes me headache

Dear Ting

I do not think that itself the lenght of your culture is a problem.

i'm routinely perform mini prep from overnight cultures in DH5 alpha growth in LB at 37°C and 180rpm and i obtain good yields.

Did you started from a single coloni?

Which culture volume? Is important to guarantee good aeration in the O/N cultures. For example i'm using 5 (max 10)ml in 50ml falcoon.

I am a little confused by your details.

You cloned into pUC19? And observe a "green" pellet?

This is the confusing part. pUC19 is not an expression vector.

Even in an expression vector you won't have green colonies.

See the attached cell pellets of a clone expressed in a pET22b system in BL21 cells. The pellet on the right is uninduced, the left is induced. We can see the GFP because I am exciting with blue light and imaging through an amber filter shield.

I would perform colony PCR to confirm size and send out for sequencing.

What media are you growing in? I would suggest Terrific Broth or 2xYT.

You also mentioned using Amp as your selection.

I would recommend using Carbenicillin or another longer lasting analog.

If the culture goes too long you can have non-clonal cells take over the mass and result in a lower yield of plasmid.

Thomas J Esparza

Re: Thank you for your response. I have cloned two plasmids. One is the plasmid that consists of GFP, while the other plasmid is pUC19

For plasmid that consists of GFP one, I have centrifugated it after overnight culture . Green pallet was found in the plasmid that consists of GFP

Manuele Martinelli Simon Flueckiger Thomas J Esparza Amy Johnson

I used 5 ml overnight culture. Yes, I promise that there is much area for the bacteria aeration. Yesterday, I have tried by using a bacterial stock that I have cloned and I have used the old mini prep kit (same brand, same apparatus with the mini prep kit that I use now) which is not prepared by myself to do mini-prep previously . It could reach a hundred or 2 hundred ng/ul. But after I bought the new mini prep kit that is same band and consists of same apparatus with my old kit, the yield of plasmid drop significantly after I cloned the same bacterial stock on the agar plate and mini-prep it by using the kit that I bought and prepared. However, I follow the protocol to mix and prepare the solution. Therefore, I am now suspecting what is happening.

Dear Amy Johnson

it is true that E.coli is facultative anaerobic but it means that it can adapt and survive in anaerobic condition for a certain time but it is not the best environment to growth and propagate it.

Oxygenation affect a lot the E.coli growth rate and its ability to reach high cell density and it is reported in many papers

Article Effect of Oxygen-Supply Rates on Growth of Escherichia coli ...

Article Oxygen availability and the growth of Escherichia coli W3110...

and recently was also observed that in anaerobic condition the E.coli has an enhanced mutation rate

Article Anaerobically Grown Escherichia coli Has an Enhanced Mutatio...

that may also explain in some way lower plasmid stability, but this was not proved it is just on my opinion. However in recombinant protein production and DNA production with E.coli is essential guarantee the right oxigenation level.

ciao

Manuele