I think, you can improve your plasmid DNA concentration by the increasing initial volume of your culture and proper incubation. I did midi-prep and maxi-prep using Qiagen plasmid extraction midi kit and megaplasmid kit and got enough plasmid DNA concentration.

Salma Mukhtar hmm, interesting, but won't it mean a higher absolute yield while kepping the concentration low?

I agree with Salma Mukhtar answer for this question

I have not a vast experience in this field, but according to my experience, for high yield or concentration of plasmid DNA, especially for sequencing purpose or restriction digestion analysis, some parameters are very important;

1. Size of plasmid or cloning vector

2. Amount of bacterial culture, e,g., 50-100 mL usually preferable for midi-prep and 250-500 mL for maxi-prep and type of bacteria (Gram-positive/Gram-negative)

3. Proper incubation at different steps during extraction process is also very important, e,g., in alkaline lysis method, after adding neutralization buffer, if mixture is incubated at ice for 30-40 min, quality may be improved.

Hopefully this information will meet your requirement.

The plasmid may be a low copy number plasmid. In the past I have been able to increase plasmid yield significantly by doing a chloramphenicol amplification. It was as simple as growing the E. coli culture overnight in the appropriate antibiotic. On the next day, add a small volume of chloramphenicol to make a final concentration of 10 to 15 ng/mL. Continue shaking for 6 to 8 hr or even overnight, and then purify DNA. You can add the chloramphenicol in addition to the regular antibiotic used for the plasmid selection (eg. ampicillin). It will not work if the plasmid carries chloramphenicol resistance. I make a 1000X stock at 15 mg/mL chloramphenicol dissolved in 100% ethanol, and add 5 uL to a 5 mL overnight. You might have to experiment with the timing.

I am assuming you did add the antibiotic matching the resistance carried by the plasmid. Because if you didn't, this would also result in little or no yield because the E. coli will not bother to keep the plasmid if there is no reason to do so.

Also, a third suggestion, if you haven't done this, call Lucigen tech support for advice. I don't mean to insult, if it seemed obvious to you, but many researchers I've known do not think of doing this, and the tech support scientists can often be very helpful. They already have experience with their product.

You can also carefully examine the genotype of the host and plasmid and see if there is anything there that is causing a problem. Again, the tech support may have suggestions.

Another situation that might apply, is to investigate whether your insert generates a product that destroys the plasmid or is toxic to the cells. A quick check to OD the overnight culture, compared to another that normally gives you good yield might show a large difference in turbidity that would correlate with low plasmid production. In that case, you will have to come up with other strategies, or do the brute force maxipreps of massive volume culture to collect the quantity you need.

Dear Itamar,

I have no idea about how much plasmid DNA you want to have for your experiment. But I can think and suggest several matter to concern. As Felise Wolven suggests, you plasmid can be low copy number. In that case you'll have to increase the volume of initial culture you use to isolate plasmids.

Second thing, always use appropriate antibiotics in prescribed concentration during the culture. Otherwise your plasmid can be easily lost from the host strain.

Thirdly, you can use lower amount of elution buffer, if you want to increase the concentration. If you used 50 ul before, now elute with 23-30 ul which will increase the concentration. After adding the elution buffer, just keep 1-2 min stand still, that also will increase plasmid concentration in the elute.

Fourth, give sufficient culturing time to your host strain (12-14 hrs). This is all coming to my mind for the moment.

All the kits you have mentioned is all good enough to have a good plasmid concentration for cloning and other experiments.

Good luck for your experiment.

Thank you.

you have some very good suggestions from Felise and also Amal. But it might help us all if you explain exactly what a "very low plasmid concentration" is. And if you tell us for what purpose the plasmid will be used.

Stupid question, but what strain of E. coli do you use? What is the volume?

The easiest way to increase the concentration (if you're somewhere close, but still want to improve the yield), is to

1) decrease the elution volume (this will decrease the total recovered DNA though, so beware. But if you're more interested in the concentration, this may really help)

2) heat the column and the elution buffer to 65-70°C before applying the elution buffer

3) let the elution buffer "sit" in the column for a minute or two. You may even try to centrifuge first for about two minutes at low speed (100 g) and then for 1 min at the regular speed.

Other plasmids work fine for you?

DH5

thank you, but after i used another kit, hiyield, i got very good concentrations