Plasmid miniprep
There are several different methods to purify plasmid DNA 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:
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Pelleting the cells by centrifuging them at 10K rpm for 3 min.
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Homogenizing the cells by adding a homogenization buffer and pipetting repeatedly.
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Lysing the cells with a lysis buffer (containing a detergent) and by inverting the tube repeatedly.
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Lysing the cells for a maximum 5 minutes at room temperature.
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Stopping the reaction by adding a neutralization buffer, and again inverting the tube several times. White clumps appear, which are the cells’ debris.
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Pelleting the debris by centrifuging for 10 min at 13K rpm. In the end, your plasmid will be in the supernatant (the liquid phase).
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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.
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Washing your DNA twice with a wash buffer (add, centrifuge, discard).
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Centrifuging 1 minute without adding anything, in order to get rid of any residual buffer.
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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.
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Checking DNA concentrations in the Nanodrop (spectrophotometer).