Keep the amp stocks frozen. On another note dh5 grows very slowly and might be the reason for low yields. I tend to use 5-10ml of overnights even for high copy plasmids this should fix your yield issues

Instead of rotating your tubes, try finding an upright incubator shaker. Vigorous mixing allows for good distribution of the cells and aeration. While E. coli does grow in low oxygen, lab strains tend to be grown with oxygen rather than in sealed tubes. I agree with the suggestion to use your entire culture volume for your plasmid prep. You can also try heating your elution buffer to 50C, and letting it sit on the column for 1 minute prior to elution. Is anyone else using the same kit? If they are also getting low yields it could be a kit issue (such as loss of ethanol in the wash buffer). Kits do expire. One last suggestion is to resuspend your cell pellet really well (vortex if you have one) until no clumps/lumps are left. Hope this helps!

Dear Emin Bursa,

despite your plasmid being a high copy number one, plasmid yield will be low if cell density (turbidity) is not adequate. You need to raise your E. coli culture in a conical flask (50-100ml flask) that permits proper shaking and aeration. From my own experience of performing mini preps and plasmid isolation, I would suggest that you grow your culture in flasks (and not in a 15ml falcon tube). A 15 ml falcon is too narrow for desired shaking and aeration, thus your OD600 will certainly be less. Secondly, you should grow your culture in a shaking incubator (Not a rototherm) at 37 degrees for at least 18 h in LB supplemented with the antibiotic. Post incubation, your culture will have a cell density that will yield appropriately sized cell pellets that would be ideal for plasmid isolation. This will certainly increase your plasmid yield. Lastly, before carrying out elution, I recommend that you pre-heat your elution buffer (transfer into a fresh MCT and place it inside a beaker filled with hot water) to ~40-50 degrees. Directly apply this buffer to your column and let it rest for 1 min and then centrifuge. This worked well for me and my colleagues and helped us greatly improve the yield of our plasmids, including pET which is a low copy number plasmid.

Wish you all the best!

As suggested by many already, it is best to use a shaker (with a back & forth movement and not Rotary) with tube holder that can be fixed at an angle to grow cultures. This helps aeration and better cell yields. Slow growth and cell yield due to rotation on a rototherm at slow speed could be an issue that you may want to address.

By any chance, does your plasmid clone harbor any insert that may not be liked by E. coli?

Another thing that we used to use way back, for maxi preps not using kits, especially for low copy plasmids, was to add chloramphenicol to the culture after it reaches reasonable cell densities (say after 5 - 6 hrs of growth). This would inhibit cell division but DNA replication would continue increasing plasmid yields.

Even though it sounds crazy, you may try the classical boiling lysis method of plasmid DNA isolation described in Sambrook et al.

For us the media caused exactly the same problem for some reason. We grew cells in 2xYT medium and the qiagen's kit yielded 300 ng/uL plasmid, meanwhile in the very same experiment LB culture (LB media was prepared from tablets) gave only 15 ng/uL. Worth a try.

After adding elution buffer I normally allow 4 minutes before I centrifuge, I also elute with 30 uL. You can try that if it helps

You could start with 2ml or in fact 3 ml culture.

Elute using 20ul.