There are factors both upstream 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 characteristics: 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, especially for plasmids ≤ 10kb. Larger plasmids, up to 25 kb, can be isolated, but yields will be reduced.
Host strain and culture conditions: The choice of host strain, antibiotic selection, growth media and culture conditions are crucial. Maintaining antibiotic selection during growth ensures that the plasmid is not lost and prevents the culture being overtaken by a faster-growing population of cells without plasmid. Proper aeration and growth temperature ensure cells grow logarithmically with little cell lysis.
Preparation procedure: Using the appropriate amount of cells and following the protocol is key. Deviating from the guidance to "boost yields" often results in reduced yield and DNA quality. Ensure the bacterial pellet is fully resuspended (no visible clumps) before adding Monarch Buffer B2 for alkaline lysis, which denatures both chromosomal and plasmid DNA.
Neutralization: Ensure neutralization is complete after Monarch Buffer B3 addition and that the plasmid DNA has renatured by checking that the solution is completely yellow before centrifugation. Incomplete neutralization may cause the cell debris (protein/ chromosomal DNA/ cell membrane) not to be pelleted to the bottom of the tube, making recovery of the supernatant more difficult. Use care to avoid transferring cell debris that may clog the Monarch column.