ü Add the entire content of supplied RNase A to the Solution Y I. Mix well. Mark the bottle label to indicate that RNase A is added. Store the buffer with RNase at 4°C.Before Starting
ü Add 1.5 volume of 96-100% ethanol to 1 volume of Wash Buffer W, Mix well. Mark on the labels that ethanol is added. Store both wash buffers with ethanol at room temperature.
ü Check the Solution Y II for precipitates. If present, warm the solution briefly at 37°C to dissolve the precipitate.
Yeast Plasmid Maxiprep Kit is designed for rapid and cost-effective small-scale preparation of high quality yeast plasmid DNA. The method is based on silica- membrane spin column.This method requires few manipulations, and is both faster and easier to perform than other organic-based extraction methods. The purified DNA is suitable for all common molecular biology procedures, including restriction digestion, cloning, sequencing, etc.
Due to the presence of a more resistant cell wall than the one present in E.coli, the plasmid DNA isolation methods commonly used for the yeast Saccharomyces cerevisiae are more difficult and produce lower DNA yields than bacterial plasmid isolation methods.
In our protocol, we performed an initial step to remove the exterior wall from yeast in order to obtain yeast protoplasts. Starting from protoplasts, we used the Yeast Plasmid DNA Purification kit to isolate plasmid DNA from yeast.
First we use lyticase to digest the wall of the yeast to obtain yeast protoplasts.Pelleted protoplasts are resuspended and subjected to SDS/alkaline lysis to liberate the plasmid DNA. The resulting lysate is neutralized to create appropriate conditions for binding of plasmid DNA on the silica membrane in the spin column . Cell debris and SDS precipitate are pelleted by centrifugation, and the supernatant containing the plasmid DNA is loaded onto the spin column membrane. The adsorbed DNA is washed to remove contaminants, and is then eluted with a small volume of the Elution Buffer (10 mM Tris-HCl, pH 8.5).The purified plasmid DNA is ready for immediate use in all molecular biology procedures such as conventional digestion with restriction enzymes, PCR, transformation and automated sequencing.
High purity: A260/A280=1.7-1.9. No chloroform and phenol, which is safe for people.
1.Transfer 1~5 ml overnight culture (~5 x 107 cells) to a sterile microcentrifuge tube.
2.Centrifuge at room temperature for 1 min at 12,000 rpm. Discard the supernatant.
3.Resuspend the cell pellet in 300 μl lyticase buffer and 50 U lyticase by pipetting up and down gently several times or by vortexing until the mixture is homogeneous. Incubate at room temperature for 30 minutes.
Note：Lyticase (Cat. # N9031/N9032) can be purchased according to your needs. The length of time required may vary for different species.
4.Centrifuge at room temperature for 10 min at 5,000 rpm. Discard the supernatant.
5.Add 250 μl Solution Y I with RNase A, resuspend the pellet by vortexing until the mixture is homogeneous. Incubate at room temperature for 1~2 minutes.
6.Add 250 μl Solution Y II. Mix gently by inverting the capped tube 5-6 times. Do not vortex. Incubate at room temperature for a maximum of 5 minutes.
Note: Vortexing will contaminate the suspension with yeast chromosomal DNA.Incubate until solution clears. Over-incubation at this stage may reduce the quality of your purified plasmid DNA.
7.Add 350 μl Solution Y III and invert the capped tube 5-6 times or until a white, free-flowing precipitate has formed. Centrifuge for 10 min at ~12,000x rpm.
8.Carefully remove and load the supernatant from step 7 onto a spin column. Incubate at room temperature for 1~2 minutes. Centrifuge for 1 min at ~12,000x rpm. Discard the flow-through.
9.Wash the column once with 500 μl Wash Buffer PB. Centrifuge at ~12,000x rpm for 1 min. Discard the flow-through.
10.Wash the column with 500 μl Wash Buffer W, Centrifuge at ~12,000 x rpm for 1 min. Discard the flow-through. Repeat Step 9 again.
11.Centrifuge the column at ~12,000x g for 3 min to remove any residual Wash Buffer W. Discard the Wash Tube with the flow-through.
12.Place the Spin Column in a clean 1.5-ml microcentrifuge tube. Add 50~100μl of preheated Eluent Buffer to the center of the column. Incubate the column for 2 minutes at room temperature. Centrifuge at ~12,000x rpm for 1 minutes. The tube contains your purified plasmid DNA. Discard the column. Store the plasmid DNA at -20℃.
Perform DNA quantitation using UV absorbance at 260 nm.
1.Prepare a dilution of the DNA solution. Mix well. Measure the absorbance at 260 nm (A260) of the dilution in a spectrophotometer (using a cuvette with an optical path length of 1 cm) blanked against the dilution buffer.
2.Calculate the concentration of DNA using the formula: DNA (μg/ml) = A260 × 50 × dilution factor
For DNA, A260 = 1 for a 50 μg/ml solution measured in a cuvette with an optical path length of 1 cm.