GENOMIC DNA PREP KIT for YEAST

ORDERING INFORMATION

Catalog #	Product	Amount	Price (EUR)
# GEN 50 Y	Genomic DNA Prep Kit for Yeast	50 preparations
# GEN 250 Y	Genomic DNA Prep Kit for Yeast	250 preparations

PRODUCT COMPONENTS

Buffer	# GEN 50 Y	# GEN 250 Y
Resuspension Buffer	6 ml	30 ml
Lyticase Buffer	75 µl	360 µl
Lyticase (final conc. 2.5 U/µl)	175 units	875 units
Yeast Lysis Buffer	20 ml	100 ml
Proteinase K (final conc. 10 mg/ml)	5 mg	5 x 5 mg
Binding Buffer	25 ml	125 ml
Column Activation Solution	6 ml	30 ml
Elution Buffer	5 ml	25 ml
Spin Columns & 2ml Tubes	50	250

DESCRIPTION

The spin column based Yeast DNA Preparation Kit is designed for rapid and high purity isolation of genomic DNA from yeast cells. The spin column based method completely removes PCR inhibitors such as divalent cations and proteins resulting in a high purity preparation of genomic DNA. There is no use of phenol or chloroform, handling is safe and does not produce any harmful waste. Column based genomic DNA purification kits yield up to 30 μg DNA sized from 200 bp to 50 kbp per preparation.

FEATURES

• Spin column type
• Purification size: 200 bp - 50 kb
• DNA amounts up to 30 µg

APPLICATIONS

• real-time PCR
  
• PCR
• southern blot analysis
  
• genotyping
• discovery or validation of SNP/SSR marker
  

BASIC PROTOCOL

Before start, prepare the following components:

• Add Lyticase Buffer to Lyticase. Lyticase Buffer containing Lyticase should be stored at -20°C.
• for 50 preparations (# GEN 50 Y): add 70 µl Lyticase Buffer to 175 units Lyticase.
• for 250 preparations (# GEN 250 Y): add 350 µl Lyticase Buffer to 875 units Lyticase.
• Add double distilled water to Proteinase K. Proteinase K solution should be stored at -20°C.
• for 50 preparations (# GEN 50 Y): add 500 µl dd-water to 5 mg Proteinase K.
• for 250 preparations (# GEN 250 Y): add 500 µl dd-water to each tube of 5 mg Proteinase K.
• Prepare an 80% Ethanol (V/V) solution as Washing buffer. Please note that the Ethanol concentration of Washing Buffer may decrease during long time storage resulting in a possible drop of the final DNA yield
• for 50 preparations (# GEN 50 Y): 60 ml
• for 250 preparations (# GEN 250 Y): 300 ml

It is essential to use the correct amount of starting material in order to obtain optimal DNA yield and purify. A maximum amount of 10⁷ yeast cells can generally be processed. Overnight cultured bacteria cells can be processed. Cell pellets can be stored at -70°C for several months.

1. Cell Lysis

• Harvest 500 µl of cultured yeast cells by centrifuge at 12,000 g for 1 min.
• Proceed immediately.
• Add 100 μl Resuspension Buffer and 1 µl Lyticase solution to the cell pellet.
• Vortex vigorously for 10 sec.
• Incubate for 15 min at 37°C.
• Centrifuge at 1,800 g for 5 min and discard supernatant.
• Add 350 μl Yeast Lysis Buffer to cell pellet.
• Vortex vigorously for 10 sec.
• Add 8 μl Proteinase K solution to the cell lysate.
• Mix by pipetting.
• Incubate at 70°C for 10 min and cool down for 5 min.
• Centrifuge for 5 min at 12,000 g at room temperature.
• Transfer supernatant to a new 1.5 ml microcentrifuge tube.

2. Adding Binding Buffer

• Add 400 µl Binding Buffer to cell lysate.
• Vortex vigorously for 30-60 sec.
• Centrifuge for 5 min at 12,000 g.

3. Column Activation

• Place a Spin Column into a 2 ml collection tube.
• Add 100 µl of Column Activation Solution into the Spin Column.
• Centrifuge at 10,000 g for 30 sec in a microcentrifuge.
  

4. Column Loading

• Pipette the lysate directly into the Spin Column.
• Centrifuge for 1 min at 12,000 g.
• Discard the flow-through.
  

5. Primary Washing

• Add 500 µl Washing Buffer (80% Ethanol) into the Spin Column.
• Centrifuge for 30 sec at 12,000 g.
• Discard the flow-through.

6. Secondary Washing

• Add 500 µl Washing Buffer into the Spin Column.
• Centrifuge for 30 sec at 12,000 g.
• Discard the flow-through.
• Centrifuge again at 12,000 g for 1 min to remove residual Washing Buffer.
  
• Discard the 2 ml wash tube and place the column in the elution tube.
  

7. Elution of DNA

• Add 40-50 μl Elution Buffer to the center of the column.
• Incubate at room temperature for 1 min.
• Centrifuge at 12,000 g for 2 min to elute DNA.
• Store DNA at 4°C (for prolonged storage, keep it at -20°C or -80°C).

STORAGE

Store at room temperature (15 - 25 °C) for 12 months.

For long term storage place Lyticase and Proteinase K lyophilisates at -20°C.

Lyticase solution and Proteinase K solution should be stored at -20°C.