There are several different methods to purifyplasmidDNA from bacterial cells. Miniprep is a rapid, small-scale isolation method, which relies on alkaline lysis of the cells, followed by silica column purification of the DNA.
The following steps have to be performed to purify plasmids from a bacteria culture:
Pelleting the cells by centrifuging them at 10K rpm for 3 min.
Homogenizing the cells by adding a homogenization buffer and pipetting repeatedly.
Lysing the cells with a lysis buffer (containing a detergent) and by inverting the tube repeatedly.
Lysing the cells for a maximum 5 minutes at room temperature.
通过添加中和停止反应buffer, and again inverting the tube several times. White clumps appear, which are the cells’ debris.
Pelleting the debris by centrifuging for 10 min at 13K rpm. In the end, your plasmid will be in the supernatant (the liquid phase).
Applying the supernatant to the silica column and centrifuging at 13K for 1 min. Your plasmid will bind to the filter at the bottom of the column. Discard the flow-through.
Washing your DNA twice with a wash buffer (add, centrifuge, discard).
Centrifuging 1 minute without adding anything, in order to get rid of any residual buffer.
Transferring your column to a clean 1.5ml tube and add elution buffer (water with 10mM Tris_HCl). Leave it on your bench for couple of minutes before centrifuging again.
Checking DNA concentrations in the nano-drop.