DNA Miniprep
The goal of a DNA miniprep is to isolate only plasmid DNA from a prokaryote. The steps are to (a) lyse the cells (b) remove cellular membranes and waste (c) wash away undesired elements and (d) elute DNA. The elution is one of the most important steps. The negatively-charged DNA noncovalently binds to a filter, and the filter is washed. Theoretically, everything will flow through except for the DNA. After washing, the DNA is then removed from the filter (a process called elution). There are several buffers commonly used to elute and store the DNA. These buffers include EB, TE and dH2O.
There are many different kits used for minipreps. All these kits follow the same steps described above. A generalized procedure for a Qiagen miniprep is:
- spin down cells
- resuspend in buffer P1
- add buffer P2; incubate
- add buffer N3; centrifuge
- run supernatant through column
- wash column with buffer PE
- elute DNA with buffer EB