User:Noah Benjamin/Notebook/Experimental Biological Chem/2011/10/05

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Miniprep/ Harvesting DNA

Using the cell culture previously grown, the liquid from the cell culture was dumped and the cells were fully resuspended using 250µL of Cell Resuspension Solution and 250µL of Cell Lysis Solution. This new mixture of cells and solution was transferred to a centrifuge tube. 10µL of Alkaline Protease solution was added to the mixture and the tube was inverted 4 times to mix contents. The mix was incubated at room temperature for 5 minutes. 350µL of Neutralization solution was added, and the tube was inverted 4 times to mix the contents. The tube was centrifuged at top speed for 10 minutes.

Liquid (NOT the solid gathered at the bottom due to centrifuging) in the tube was pipetted into a "spin column" and was vacuumed through the spin column using a Vaccum Adapter and a Manifold Port (specialized instrumentation).

750µL of Wash Solution (ethanol added) was used to wash the contents of the spin column by applying vacuum. This was repeated using 250µL of the Was Solution. The contents of the spin column were dried by vacuuming for 10 minutes. The column was placed inside a 2 mL collection tube, filter-side down, and was centrifuged at top speed for 2 minutes.

The column was transfered to a sterile 1.5mL centrifuge tube. 100mL of Nuclease-Free Water was added to the column, and the column was centrifuged at top speed for 1 minute. The column was discarded and the contents of the centrifuge tube were labelled and stored at -20 degrees Celsius.


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