DNA preparation by pulsed-field gel electrophoresis (isolation of yeast intact DNA)
DNA preparation by pulsed-field gel electrophoresis (isolation of yeast intact DNA)
To prepare yeast DNA for electrophoresis, the cell walls of yeast cells are enzymatically disrupted and then suspended in melted low melting point agarose to make a bolus. The bolus is immersed in a lysis buffer containing protease to accelerate cell lysis and remove proteins. This method is a slight modification of the one originally described by Schwartz and Cantor (1984), which can be used to prepare yeast chromosomes and yeast artificial chromosomes (YACs) as polymer quality standards. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Peitang Huang et al.
Operation method
DNA preparation by pulsed-field gel electrophoresis (isolation of yeast intact DNA)
Principle
To prepare yeast DNA for electrophoresis, the cell walls of yeast cells are enzymatically disrupted and then suspended in melted low melting point agarose to make a bolus. The bolus is immersed in a lysis buffer containing protease to accelerate cell lysis and remove proteins. This method is a slight modification of the one originally described by Schwartz and Cantor (1984), which can be used to prepare yeast chromosomes and yeast artificial chromosomes (YACs) as polymer quality standards.
Materials and Instruments
Yeast Suspension Cultures Move I. Materials For more product details, please visit Aladdin Scientific website.
Cell Wash Buffer EDTA L Buffer TE Yeast Lysis Buffer Yeast Cell Wall Digestive Enzyme Enzymes
Low melting point agarose Sorvall GSA Turning head or equivalent Pre-formed Plexiglas molds
1. Buffers and solutions
Cell wash buffer (0.01 mol/L Tris-Cl ( pH 7.6), 0.05 mol/L EDTA ( pH 8.0), stored at room temperature.)
EDTA ( 0.05 mol/L, pH 8.0)
L buffer (0.1 mol/L EDTA (pH 8.0), 0.01 mol/L Tris-Cl (pH 7.6), 0.02 mol/L NaCl, stored at 4°C)
L buffer with proteinase K and sodium dodecyl sarcosinate (Sarkosyl)
TE (pH 7.6)
TE containing 40 μg/ml PMSF (pH 7.6)
Yeast lysis buffer (0.01 mol/L Tris-Cl (pH 7.6), 0.5 mol/L EDTA (pH 8.0), β-mercaptoethanol (1% V/V))
Yeast cell wall digestive enzymes
Zymolyase 5000 (Kirin Breweries) or Lyticase (67 mg/ml) (Sigma)
2. Gel
Low melting point agarose (1%)
3. centrifuges and rotors
Sorvall GSA turntable or equivalent
4. Specialized equipment
Pre-formed Plexiglas mold (50-100 μl, Pharmacia or Bio-Rad), or a long Tygon tube (1/8 inch or 3.2 mm ID), or a plastic syringe (1 ml).
5. Cells and tissues
Yeast mixed suspension cultures
II. Methods
1. 3000 g ( Sorvall GSA turntable 4300 r/min) 4°C centrifugation for 5 min Collect yeast cells from suspension culture. Wash the cell sediment twice with cell wash buffer.
2. Suspend the cells in 0.05 mol/L EDTA (pH 8.0) at 0℃ at a concentration of 3X109 cells/ml.
3. Prepare 1% low melting point agarose in L buffer. Melt and cool to 42°C.
4. Add 75 μl of enzymatic or cytolytic enzyme solution to the cell suspension from step 2 and mix well.
5. Heat the cell suspension to 42°C. Mix 5 ml of melted agarose with 5 ml of cell suspension. Stir the mixture with a closed Pasteur pipette to ensure that the cells are completely dispersed in the agarose.
6. Transfer the mixture into a pre-formed Plexiglas mold (50-100 μl, Pharmacia or Bio-Rad), or pipette the mixture into an appropriate length of Tyson tubing (3/32" ID), or into a 1 ml plastic syringe. Leave at room temperature for 15 min, then move to 4°C for 15-30 min.
7. After the agarose has solidified, collect the gel plug from a Plexiglas die or squeeze the gel from a Tygon tube and syringe. Cut the cylindrical gel plugs into 1 cm long pieces.
8. Incubate gel blocks in 3x volume of yeast lysis buffer at 37°C for 3 h in a chemical fume hood.
9. Add 3x volume of L buffer containing 0.1 mg/ml Protease K and 1% (m/V) Sarkosyl to a clean petri dish. Transfer to the gel block and incubate at 50°C for 3 h. Replace the old buffer with a fresh volume of the above buffer and continue to incubate at 50°C for 12-16 h.
10. Incubate the gel block in a 50-fold volume of TE (pH 7.6) containing 40 μg/ml PMSF at 50 °C for 1 h. Replace the old buffer with a fresh volume of the above buffer and continue to incubate for 1 h at 50 °C.
11. Remove the PMSF-containing TE and continue to incubate for 1 h at room temperature with a fresh volume of TE (pH 7.6).