Dos and Don'ts of Plasmid Minipreps

DON’T use too many cells
If the recommended amount of cells is exceeded, the amount of Monarch^® Buffer B2 recommended in our Monarch Spin Plasmid Miniprep Kit protocol may not be able to efficiently lyse all the cells. Also, excess cell debris resulting from lysis of too many cells can clog the column. If you need to use more cells than recommended, consider splitting the sample in half and using two columns.

DO lyse your cells completely
In order to release ALL of the plasmid DNA, ALL of the cells need to be lysed. To do this, make sure the cells are resuspended completely, without any clumps, and incubate the cells for the recommended amount of time.

DON’T vortex your cells after lysis
Vortexing can cause shearing of host chromosomal DNA, resulting in gDNA contamination.

DON’T skip or shorten the RNase A digestion step
Make sure to add provided RNase A to Monarch Buffer B1 before resuspension. It's important to follow all incubation recommendations to ensure complete RNA removal, especially after addition of Monarch Buffer B3, as this is the time when RNase A digests the contaminating RNA.  If >2.5 ml of cell culture is used, increasing the spin time after neutralization to 5 minutes will help.

DO follow wash steps as directed
Both Monarch Buffer BZ and Monarch Buffer WZ should be used in the volumes recommended to ensure removal of cell debris, endotoxin and salts.

DON’T mix up your buffers
The buffers need to be added in a particular order, since each one carries out a different function in the purification workflow. Using them out of order can cause your miniprep to fail. Monarch miniprep buffers are color coded for your convenience.

DON’T let the tip of the column touch the flow-through in the collection tube after washing
Ethanol can carry over from the collection tube to the column tip. Ethanol in your eluate can interfere with downstream applications. If you suspect that the tip has touched the flow-through, another spin should do the trick.

DO heat the elution buffer when purifying large plasmids (>10 kb)
Large DNA binds more tightly to the silica matrix. Heating the Monarch Buffer EY before applying to the column helps to more efficiently release the DNA from the matrix.