Low copy numbers of plasmids

Question

I have a plasmid with the P15A origin which apparently has a low copy number (see here). This would explain why my purification yeilds for subsequent digestion are low (gel shows the plasmid after digestion and clean-up with 5µl being loaded). I am currently growing my E. coli containing plasmid up in 5mls overnight before doing a miniprep. My question is should I grow my 5ml cultures up for longer (2 days) or just give it up and do a larger amount for Midiprep.

    

I am also curious as to what actually controls the plasmid copy number inside cells. An explanation of the regulation of plasmid copy number would be much appreciated.

Answer 1

• I am sure you have done it; just a note to others: do a PCR screen to ascertain that the plasmid is there.
• For minipreps you generally do not need a starter culture.
• If you have screened your clones and now just want to amplify and keep the plasmid for cloning purposes then go for a maxiprep with 200-400ml cultures.
• Do not overgrow the cells, your extraction becomes difficult and you would get even worse yields. Just set up a huge culture and pool the cells before extraction. Always seed and harvest cells that are in exponential growth phase.
• If you need to screen the plasmid for clones or something then do a colony PCR, keep the colony inoculated in a small culture (make a temporary glycerol stock if your screening is taking time). If screen is positive then incoulate the starter culture for maxiprep/gigaprep culture with this stock.