Hi Jafeth,

How much of bacterial culture are you using to do a plasmid miniprep ???

Try using a higher volume of bacterial culture to start with, like 20-30 ml. That would increase the plasmid concentration.

You could also concentrate the plasmid samples together from different minipreps and get a higher concentration. Try eluting in only 30-35 ul of TE, so that the concentration is higher and you would only need a small volume of miniprep for your cloning experiment.

Hello

It is usual for most extraction protocols to use from 30 microliter of elution buffer instead of 50 microliters. In this way, your DNA concentration would be increased by several orders

My plasmid is from Addgene with high copy number in E. coli Dh5 alpha. I have eluted 10 ul x2 and still have low concentrations. I have inserted one more washing step as well. I have not tried heating the elution buffer I have used from 1.5 to 10 ml of overnight culture (16 hours, but I have never measured DO and it seems like a clear culture). I have used 50 ug/ml to 100 ug/ml ampicilin

Are your columns fine? Or is the problem with your plasmid? Try mini prep of some empty vectors or some positive clones, which have worked fine earlier. 5ml culture is more than enough, and should give yield in micrograms (total).

I followed all the advice you gave me and tried again to purify the plasmid with my kit and tried with a promega kit. Using 4.5 ml of culture with my kit I recovered 5.1 ng / uL and with the promega kit I recovered 119.6 ng / ul.

Looks like it was a kit issue. If so, it may be time to dispose of the Bioland Scientific kit! Did you notice any differences along the day, perhaps regarding cell lysis or lysate clearing?

Jafeth, then it's clearly column issue. Write to your supplier regarding defective supply