Purification of Plasmids from Yeast and Bacterial Transformation
Plasmid Purification from Yeast
In this step you will extract DNA from yeast and transform it into E. coli. You will first perform enzymatic lysis of the yeast cells using the enzyme Zymolyase. The goal is to remove the tough cell wall of the yeast cells and release the DNA into solution. You will then precipitate the cellular debris using standard alkaline lysis (first solubilize the cell membranes using SDS, then cause them to flocculate using potassium acetate) and pellet the cellular debris by centrifugation. Finally, you will pass the supernatant over a column to which the DNA will bind. Following a wash step, the DNA will be eluted in a small volume.
1. Add 2 ml sterile water to the yeast transformation plate and scrape off the colonies from the plate with a sterile wooden stick.
2. Using a P1000 pipet set to 1000, transfer the cell slurry into a microcentrifuge tube.
3. Spin down the cells at top speed for 30 seconds and remove the supernatant with a pipette.
4. Resuspend pelleted yeast cells in 250 μl TE buffer. Add 50 μl of Zymolyase solution.
5. Add 300 μl Solution 2 and mix thoroughly by inverting the tube 6–8 times to mix. The solution should become viscous and slightly clear. Do not vortex.
6. Add 300 μl Solution 3 and mix immediately and thoroughly by inverting the tube 6–8 times. The solution should become cloudy.
7. Centrifuge for 5min at top speed at room temperature.
8. Transfer the supernatant into a spin column that is placed in a collection tube. Don’t forget to label the spin column directly with the name of the chunk.
9. Centrifuge at top speed for 60 seconds.
10. Discard the flow through in the collection tube and replace the spin column into the empty collection tube.
11. Add 200ul Endo Wash buffer and centrifuge 1 min. Discard flow through.
12. Add 400uL Zyppy wash buffer onto the spin column and centrifuge at top speed.
13. Discard the through in the collection tube and spin 1 more time to remove residual ethanol. Place the spin column into a fresh eppendorf tube.
14. Add 30uL of elution buffer directly onto the column.
15. Incubate for 1-2 minute at room temperature.
16. After verifying that you have place the spin column into a clean eppendorf (as described in step 13), centrifuge at top speed for 1 minute.
17. Your DNA will elute into the eppendorf tube. Discard the spin column.
Bacterial Transformation of Assembled Plasmids
We have assembled the RNR3 promoter, GFP gene, and a vector both by Gibson assembly and by yeast assembly. We now must transform the assembled RNR3-GFP construct into bacterial. This serves two functions. First, bacteria can be stored long-term, allowing us to create a permanent stock of the RNR3-GFP construct. Second, bacterial transformation is also relatively inefficient-this means that each bacterial cell will contain only one plasmid (a clone), which we can screen to determine if it contains a perfect copy of the RNR3-GFP construct.
You will be performing four reactions:
- Transformation of the RNR3-GFP gene assembled in yeast
- Transformation of the RNR3-GFP gene assembled by Gibson assembly
- A positive control (a plasmid that should transform bacteria at high efficiency)
- A negative control (water; this should not transform bacteria unless there is contaminating DNA present)
1. Obtain 4 tubes and label with your initials and numbers 1-4. Prechill the tubes by placing them on ice.
2. To each tube, add 2 ul of the appropriate DNA (see description of the four reactions above).
3. To each tube, add 100 ul of chilled bacterial culture in TSS*. Flick the tube gently.
4. Incubate on ice 30 min
5. Add 900 ul of SOC media
6. Incubate at 37C for 1 hour
7. Spin down tubes and remove all of the liquid, leaving the bacterial pellet intact.
8. Resuspend the bacteria in 100 ul of water, pipetting up and down gently until the suspension is smooth with no clumps of cells.
9. Pipet the transformed bacteria onto the center of an LB+Amp plate. Spread the bacteria on the plates.
10. Incubate the plates at 37C. Growth of colonies should be apparent after ~16 hours.
- Note: TSS consists of LB broth with 10% (wt/vol) PEG (molecular weight 3350 or 8000), 5% (vol/vol) DMSO, and 20-50 mM Mg2+ (MgSO4 or MgCl2), at a final pH of 6.5