HomeFAQsI have used 5 ml of cell culture for plasmid isolation with the Monarch Plasmid Miniprep kit and I am obtaining low amounts of plasmid and/or contaminating gDNA. Why is this, and what are your suggestions to improve yield and purity?
FAQ: I have used 5 ml of cell culture for plasmid isolation with the Monarch Plasmid Miniprep kit and I am obtaining low amounts of plasmid and/or contaminating gDNA. Why is this, and what are your suggestions to improve yield and purity?
A lower yield when using 5 ml cell cultures may indicates that too many cells were used in the plasmid isolation. Though we advise that our kit can accommodate up to 5 ml of culture, there are some cases in which the density of the cells is too high with this volume. Using too many cells can result in incomplete cell lysis, reduced removal of host gDNA, and lower yield due to an insufficient volume of Monarch Lysis Buffer (B2) used in the standard protocol. Additionally, the lysis of too many cells will produce excess cell debris and contaminating gDNA that can overwhelm the column and decrease its capacity for binding the plasmid DNA. If the cell culture is being harvested after it has transitioned to the stationary phase, then using less cell culture volume in the isolation may provide better yields. Splitting the 5 ml culture in half and processing on two columns will likely produce better results. Alternatively, doubling the resuspension, lysis, and neutralization volumes may allow better column performance but will necessitate loading the column twice, each time with half the neutralized sample.
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