FAQ: What factors affect recovery of plasmid DNA?

There are factors both upstream of and during the prep that can affect plasmid recovery.  Plasmid copy number, plasmid size, insert toxicity, host strain, antibiotic selection, growth media, and culture conditions can all affect downstream plasmid recovery. Plasmid copy number, size, and insert toxicity directly affect the amount of plasmid contained in each cell. For higher yields, choose high copy plasmids when appropriate and keep total size ≤ 10kb. Larger plasmids, up to 25 kb, can be isolated but yields will be reduced. Maintaining antibiotic selection during growth also ensures plasmid is not lost during growth and that the culture is not overtaken by a faster growing population of cells without plasmid. Proper aeration and growth temperature ensure cells grow logarithmically with little cell lysis. 

During the preparation, using the appropriate amount of cells and following the optimized protocol ensures efficient alkaline lysis will occur and the column will not be overloaded. Deviating from the guidance provided to "boost yields" often results in reduced yield and DNA quality. Ensure the bacterial pellet is fully resuspended (no visible clumps) prior to adding buffer B2 for alkaline lysis, which denatures both chromosomal and plasmid DNAs.  Also, ensure neutralization is complete after buffer B3 addition and that the plasmid DNA has renatured by checking that the solution is completely yellow prior to centrifμgation to clear the lysate. Incomplete neutralization may cause the cell debris  (protein/ chromosomal DNA/ cell membrane) to not be pelleted to the bottom of the tube, making recovery of the supernatant more difficult.  Use care not to transfer any of the cell debris to the Monarch column to avoid clogging.