Are you planning to perform some plasmid minipreps? Don’t stress! Here are some basic things to keep in mind in order to get clean plasmid DNA, ready for use in downstream applications.
DON’T use too many cells
If the recommended amount of cells is exceeded, the amount of lysis buffer recommended in our Monarch® 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
Lucky for you, Monarch Neutralization Buffer comes with RNase A already added (other kits require you to add it - an extra step that is easy to forget!). The neutralization step is very important, as this is the time when RNase A digests the contaminating RNA. It is important to follow the incubation recommendations for this step to ensure complete RNA removal. If >2.5 ml of cell culture is used, increasing the spin time after neutralization to 5 minutes will help.
DO use both wash buffers as directed
Both Monarch wash buffers 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 elution buffer before applying to the column helps to more efficiently release the DNA from the matrix.