Yeast plasmid extraction centrifugation procedure

Yeast plasmid extraction centrifugation procedure:

1. Inoculate 5 mL of YDP medium containing the plasmid required for yeast carrying, and shake it at 30 ° C for 16-24 hours.

2. Take 1-3 ml of yeast cultured cells (using < 2 x 107 cells) and centrifuge at 5000 x g for 5 min at room temperature.

3. Discard the culture medium, collect the cells, and add 480 μL Buffer SE and 30 μL Lyticase solution . Vortex for 1 min at the fastest speed. Full suspension of cell pellets helps increase yield. It was digested at 30 ° C for about 30 minutes.

Note: Make sure that β-mercaptoethanol has been added to Buffer SE (10 μL / ml) before use. This mixture can be well placed at room temperature for 1 week.

4. Centrifuge the mixture pellets at 4,000 xg for 5 min at room temperature and discard the supernatant completely.

5. Resuspend the mixture particles by adding 250 μL Buffer YPI .

6. Add 50 mg glass beads and vortex for 5 min at the fastest speed to allow the sample to settle under the action of glass beads. Transfer the supernatant to a 1.5 ml centrifuge tube.

7. Add 250 μL Buffer YPII and mix the tube 4-6 times by inverting to obtain a clean dissolved product. Leave it at room temperature for 5 min.

Note: This step should avoid high-intensity mixing as this will break the chromosomal DNA and reduce the purity of the plasmid. YPII should cover the lid when storing.

8. Add 350 μL of Buffer YP III and mix the sample thoroughly by hand until a flocculated white precipitate forms. Centrifuge at 13,000 × g for 10 min at room temperature.

9. Carefully transfer the clean supernatant to the DNA Mini Column and try not to disturb the particles and precipitate when removing the supernatant. Centrifuge at 10,000 × g for 30 s at room temperature. Pour off the waste and reinsert the column into the collection tube.

10. Add 500 μL of Buffer KB to the column and centrifuge at 12,000 rpm for 30 seconds to discard the collection tube and waste.

11. Place the column into a new collection tube and add 650 μL Wash Bufffer (ensure that ethanol has been added), centrifuge at 12,000 for 30 seconds, drain the waste, and reinsert the column into the collection tube.

Note: Wash Buffer is diluted with absolute ethanol and used.

12. Optional step: Repeat step 11.

13. Centrifuge at 13,000 rpm for 2 minutes.

Note: Open lid centrifugation helps remove residual ethanol, and efficient removal of ethanol ensures DNA elution.

14. Place the column in a new 1.5 ml centrifuge tube, add 50-100 μL of Elution Buffer (10 mM Tris-HCL, pH 8.5) to the column, allow to stand at room temperature for 1 min, and centrifuge at 13,000 xg for 1 min.