This video protocol shows you how to perform an alkaline lysis plasmid miniprep. The DNA purification industry would like you to believe that their kits are necessary to obtain plasmid DNA suitable for routine applications. This is simply not true. In fact, isolating large quantities of high purity plasmid DNA is really easy. Plasmid miniprep procedures commonly use columns that give low yield and are relatively expensive. This modified version of the traditional alkaline lysis miniprep produces large yields of high quality DNA and costs less than $0.05 per prep (including cost of tubes). Download a printable plasmid miniprep protocol here. The protocol described here (and shown in the video) has a few modification from the classical miniprep protocol found in books such as “Molecular Cloning” that increase the purity of the plasmid DNA.
Advantages of this miniprep method
- Large yield
- High purity
- Fast
- Cheap, 20 times less than most commercial kits.
Tips
- The hole in the cap of the tube can be taped shut after cultures have finished growing, when using 2 mL microcentrifuge tubes to grow cultures. Pelleted E. coli can be frozen at -20 degrees C after the supernatant is discarded and processed later.
- It is important not to transfer and white precipitate to the new tube at step 8 in the protocol. However, you will have a second chance to get rid of the white precipitate later. When resuspending the ethanol pellet in Tris buffer (step 13) you may have some white precipitate that does not resuspend. This material is not DNA. Therefore, you can spin the insoluble material to the bottom of the tube and transfer the clear liquid to a new tube.