Products

Yeast HighPure Plasmid Extraction Maxi Kit(SpinColumn)

Packing Specification：

Product Introduction:
This kit uses an improved SDS-alkaline lysis method combined with cell wall-breaking enzyme for specific digestion of yeast cell walls, enabling the isolation of high-purity plasmid DNA from yeast cultures within 1 hour. After yeast collection, cell walls are removed by adding cell wall-breaking enzyme, followed by cell lysis via alkaline lysis. The silica matrix membrane in the spin column selectively binds plasmid DNA in the solution under high-salt and low-pH conditions. Impurities and other bacterial components are removed through deproteinization solution and wash buffer. Finally, pure plasmid DNA is eluted from the silica matrix membrane using low-salt and high-pH elution buffer.

Application Scope:
Suitable for large-scale preparation of high-purity yeast plasmids.

Product Features:
1.The silica matrix membranes in the spin columns are all specially manufactured adsorption membranes imported from world-renowned companies, ensuring minimal variation in adsorption capacity between columns and good reproducibility. It overcomes the drawback of unstable membrane quality in domestic kits.
Rapid and convenient: Eliminates the need for toxic reagents such as phenol and chloroform, as well as ethanol precipitation. The obtained plasmid has high yield and purity, and can be directly used for various molecular biology experiments such as restriction digestion, transformation, PCR, in vitro transcription, and sequencing.

Operation Steps:
Tips:
Before first use, add the specified volume of absolute ethanol to the Wash Buffer WB bottle, mix thoroughly. Mark the corresponding box immediately after adding ethanol to avoid repeated additions!
Add all RNase A to Solution YP1, mix well, and store at 2-8℃ after each use.
Precool Solution YP3 on ice.
Add 0.1% β-mercaptoethanol to the required volume of Sorbitol buffer and bring to room temperature for later use.
1.Add approximately 120-200 mL of yeast culture to a 50 mL centrifuge tube, centrifuge at 8,000 x g (approximately 9,000 rpm) for 1 minute, pour off the supernatant as much as possible, and collect the bacterial pellet.
For collecting more than 50 mL of bacterial culture: Centrifuge to discard the supernatant, add more bacterial culture to the same 50 mL tube, and repeat Step 1 until sufficient bacterial pellet is collected.

2.Add 10 mL of Sorbitol buffer, gently pipette to fully resuspend the cells; add 0.1 g of cell wall-breaking enzyme (dissolve the enzyme in 2 mL of Sorbitol buffer before use), invert thoroughly to mix, incubate at 37℃ for 1-2 hours to digest the cell wall, and invert several times during incubation to assist digestion.
If poor cell wall breaking leads to low plasmid yield: Increase the dosage of cell wall-breaking enzyme to improve enzyme working concentration, extend digestion time, or increase temperature to 45℃. For yeasts not suitable for cell wall-breaking enzyme digestion, Lyticase, Zymolase, or other methods such as vortexing with glass beads or repeated freeze-thaw can be used.

3.Centrifuge at 8,000 x g (approximately 9,000 rpm) for 2 minutes, aspirate and discard the supernatant as much as possible, add 7 mL of Solution YP1 to resuspend the bacterial pellet, vortex until completely suspended.
Undissolved bacterial clumps will affect lysis, leading to low yield and purity.

4.Add 7 mL of Solution YP2, gently invert up and down 6-8 times to fully lyse the bacteria, and incubate at room temperature for 4 minutes.
Mix gently; do not shake vigorously to avoid shearing genomic DNA! The incubation time should not exceed 5 minutes to prevent plasmid damage. The bacterial solution should become clear and viscous at this time. If there are few bacteria, proceed to the next step once it becomes clear and viscous; exact 5-minute incubation is not required. If the solution does not become clear, it may be due to excessive bacterial biomass and incomplete lysis; reduce the bacterial amount accordingly.

5.Add 10 mL of precooled Solution YP3, immediately invert up and down gently 6-8 times. White flocculent precipitation will form when fully mixed. Incubate on ice for 5-10 minutes, centrifuge at 8,000 x g (approximately 9,000 rpm) at 4℃ for 2 minutes, carefully transfer the supernatant, and avoid aspirating the floating white precipitate.
Mix immediately after adding Solution YP3 to avoid local precipitation of SDS. If there are still floating white precipitates in the supernatant, centrifuge again and transfer the supernatant.

6.Load the supernatant obtained in the previous step into the Spin DC Adsorption Column (placed in a collection tube), let stand for 2 minutes, centrifuge at 8,000 x g (approximately 9,000 rpm) for 1 minute, and discard the waste liquid in the collection tube.
In cases where the centrifuge rotor has a large inclination angle, it is recommended to load no more than 9 mL of solution into the adsorption column each time to prevent leakage. If the supernatant volume exceeds 20 mL, load in multiple batches until all the mixture has passed through the adsorption column.

7.Add 10 mL of Deproteinization Buffer PE, centrifuge at 8,000 x g (approximately 9,000 rpm) for 1 minute and discard the waste liquid.

8.Add 10 mL of Wash Buffer WB (check if absolute ethanol has been added!). Centrifuge at 8,000 x g (approximately 9,000 rpm) for 1 minute and discard the waste liquid. 

9.Repeat Step 8 once.

10.Place the empty adsorption column back into the collection tube, centrifuge at 8,000 x g (approximately 9,000 rpm) for 3 minutes to dry residual ethanol on the matrix membrane. Aspirate any residual ethanol between the inner ring pressure ring and the column wall with a pipette tip, then air-dry at room temperature or in an oven for a few minutes.
This step is to completely remove residual ethanol in the adsorption column, as ethanol residue inhibits downstream reactions and significantly reduces elution efficiency and plasmid yield.

11.Transfer the Spin DC Adsorption Column to a clean 50 mL centrifuge tube, air-dry at room temperature for 2-3 minutes. Add 1 mL of Elution Buffer EB (preheating the elution buffer in a 65-75℃ water bath can improve yield) to the center of the adsorption membrane. Incubate at room temperature for 2 minutes, centrifuge at 8,000 x g (approximately 9,000 rpm) for 1 minute, and discard the adsorption column.
To increase plasmid recovery efficiency, reapply the eluate to the spin column, incubate at room temperature for 1 minute, and centrifuge at 8,000 x g for 1 minute. Eluting twice can increase the concentration by approximately 10%.
Higher elution volume leads to higher elution efficiency. To obtain higher plasmid concentration, the elution volume can be appropriately reduced, but the minimum volume should not be less than 0.6 mL (excessively small volume reduces plasmid elution efficiency and yield).