Is easy to say protocol was followed according to the manufacturer, several steps in plasmid preps can impact the eventual yield, pls watch the cell lysis step, equilibration of the column, clarification of lysate- impurities in the column could be problematic, elution ( do you do a short bench top incubation before elution?). Goodluck as you figure out where exactly to improve the yield of your plasmid prep.

Did you make starter culture before growing the cells in the 4*500 media?

If no, I would suggest to do make starter culture first, leave it overnight and then transfer to the 4*500mL.

So the sequence would be:

Transform, Plate on agar, Colony PCR for insert confirmation, inoculate in starter culture, transfer an overnight starter culture to the larger volume media.

Use the attached protocol for getting the high efficacy

Andrew Ting Thank you for your answer. Yes, after transformation, I've plated bacteria on agar (with antibiotics), then picked up 1 colony and inoculated in 5mL of LB antibiotic, cultured overnight and verified the profile by restriction enzymes after miniprep extraction of the plasmid. Finally, I used these starter culture to inoculate the 4*500 mL media.

Something that can aid DNA and RNA extraction in general is checking your buffers. Are they old? Did you use molecular grade or high quality ethanol? If you are using a 70% EtOH solution in any of your steps, make sure it is fresh every single time. I also second the advice to do an incubation step during elution. Another place you could add an elution step is to let you lysed solution sit on the column for a few minutes before running it through.

I have never used that kit before, but I have had great success in the past using the Qiagen version.

I don't think DNA concentration of 1-2mg is low. This is equivalent to 1000ng which is surely OK for you to do any downstream application.

Haleigh Conley I'm using a new kit so all the buffers are new and freshly prepared (

Laury-Anne Leroy, even though your column may have up to that capacity but the copy number of your plasmid is equally important. I believe by getting 1-2mg, your plasmid is a high copy number plasmid. The highest concentration I ever got was 400ng by using XL-ultragold competent cells from agilent technologies. So no matter how much you try to get higher concentration from that , I'm not sure if that will be possible because that's probably the capacity of the plastic you're using. You can goggle and see the capacity of various high copy number plasmids and let see if you can get one that can yield up to that amount