Why is the yield of my plasmid prep so low?
The origin of replication on your plasmid is for low-copy replication
The number of plasmid copies per bacterial cell is determined by the origin of replication on the plasmid. Some origins have inherent low copy number. Check the copy number of the origin of replication on your plasmid. For low-copy plasmids, increase the amount of E. coli culture for plasmid DNA prep in order to obtain satisfying DNA yield.
The volume of bacterial culture is too low for plasmid prep column
Please check the binding capacity of your plasmid prep column and whether your plasmid is high- or low-copy plasmid. For mini preps, we recommend that you harvest 1-5 ml of overnight bacterial culture. For maxi preps, if the plasmid is high-copy plasmid, we recommend using 100-150 ml of overnight bacterial culture; if the plasmid is low-copy plasmid, we recommend using 300-500 ml of overnight bacterial culture. Typically, for high-copy plasmid, ~5 ug of plasmid DNA can be extracted from every 1 ml of culture in mini prep and ~500 ug of plasmid DNA can be obtained from 150 ml of culture; for low-copy plasmid (e.g. pET), 1.5-2.5 ug of plasmid DNA can be harvested from every 1 ml of culture in mini prep and 150-200 ug of plasmid DNA can be obtained from 150 ml of culture.
Only a fraction of bacteria in the liquid culture contain plasmids
Some antibiotics, ampicillin in particular, degrade fast in liquid culture. As a result, bacteria that do not contain plasmids can propagate to a significant fraction of the culture, causing poor yield of the plasmid prep. To avoid this, please prepare ampicillin containing growth medium freshly before use and make sure that enough ampicillin is supplied. Also, when culturing ampicillin-resistant bacteria, do not let the liquid culture saturate for too long before harvesting. Besides insufficient antibiotics in the culture, extracting plasmid DNA from very old culture can also result in low yield, since many bacterial cells are dead and plasmid DNA they contain is degraded. Therefore, try to extract plasmid DNA from fresh culture. If plasmid prep cannot be performed immediately, you can spin down the bacteria and store the pellet in -80°C freezer for later plasmid prep.
The liquid culture is directly inoculated from E. coli stab culture
Direct inoculation of a liquid culture from the E. coli liquid stock or stab culture you have received from VectorBuilder can very occasionally result in low yield. We recommend streaking the stock onto an LB agar plate containing the appropriate antibiotic first, and then inoculating a liquid culture with a fresh colony growing from that plate. Detailed user instructions can be found by going to menu item Learning Center > Documentation > Stab Culture.
You have not carefully followed the manual of the plasmid prep kit
If you use a plasmid prep kit, please carefully read the manual before use. Improper operations can often lead to poor performance of the kit.
The mini/maxi prep column is low-quality
Some brands of plasmid DNA prep columns perform poorly or inconsistently for DNA preparation.