Obtain the plasmid-bearing bacterial cells
1. Collect the three bacterial cultures that your group has been assigned. The bacteria have been transformed with plasmids containing either an S. cerevisiae MET gene, its S. pombe ortholog or bacterial LacZ. Each culture contains 0.6 mL Luria Bertani (LB) medium with 100 μg/mL ampicillin. Cultures were grown overnight at 37 degrees C, and the cell density is expected to be 3-4 X 109 cells/mL.
What is the purpose of the ampicillin? How does it work?
Alkaline lysis of bacterial cells harboring the plasmids
2. Add 100 μL of 7X Blue Zyppy Lysis buffer to the tube. Mix the buffer and cells by gently inverting the tube 4-6 times. Be gentle! Too much mechanical stress could fragment the bacterial chromosomal DNA and contaminate your plasmid preparation. The solution should turn from a cloudy blue to a clear blue.
NOTE: This step is time-sensitive!! Proceeed to the next step within 2 minutes.
Separate plasmid DNA from denatured proteins and chromosomal DNA
3. Add 350 μL of cold Yellow Zyppy Neutralization buffer (w/RNAase A) to the tube, and mix the contents thoroughly by inverting several times. The solution will turn yellow when neutralization is complete, and a yellowish precipitate will form. Invert the sample an additional 3-4 times to ensure complete neutralization. Be sure there is no more blue color.
4. Centrifuge the mixture at maximum speed for 3 minutes to remove denatured proteins and chromosomal DNA. Notice that the tube contains a white precipitate that has collected on one side of the tube. The pale yellow supernatant contains the plasmid DNA.
Purify plasmid DNA by adsorption to a silica resin.
5. Using a pipette, carefully transfer the pale yellow supernatant (~900 μL) onto a Zyppy spin column. Be careful not to transfer any of the yellow precipitate! Label the column - NOT the collection tube that you will use in the next step.
6. Place the column with the collection tube attached into a centrifuge and spin at maximum speed for ~ 1 minute.
7. Remove the column and discard the flow through in the collection tube.
8. Place the column back into the collection tube and add 200 μL of Zyppy Endo-Wash solution. (Endo-Wash contains guanidine hydrochloride and isopropanol, which will remove denatured proteins from the resin.)
9. Centrifuge for ~1 minute and discard the flow through.
10. Place the column back into the collection tube then add 400 μL of Zyppy Column Wash buffer. (This steps removes contaminating salts.) Centrifuge for ~1 minute. Empty the collecting tube.
11. Repeat the centrifugation step to remove any residual ethanol.
Elute the plasmid DNA
12. Transfer the Zyppy column to a clean (and appropriately labeled) 1.5 mL centrifuge tube, leaving the lid of the tube open.
13. Carefully, add 100 μL of elution buffer directly on top of the white column bed. Place the pipette tip as close as you can to the white column bed without poking it. Slowly dispense the buffer on top of the resin bed.
14. Allow the buffer to percolate into the column by letting the column sit in the microcentrifuge tube for 1 minute.
15. Centrifuge the column at maximum speed for 30 seconds. Again, it’s fine to leave the cap open during this spin.
16. Remove the column, cap the tube and place it on ice. This tube should now contain plasmid DNA. Label the tube. SAVE the DNA for future experiments.