Yeast DNA Preparation - Column Kit

For general laboratory use.

Shipping: shipped at ambient temperature

Storage Conditions: store at ambient temperature

Shelf Life: 12 months

Applications:
The obtained DNA is suitable for a variety of applications, including real-time PCR, southern blot analysis, genotyping and discovery or validation of SNP/SSR markers.
Before start, add the following components as indicated on the respective bottle/tube:

• Lyticase Buffer to Lyticase
• double distilled water to RNase A and Proteinase K
• 96-99 % Ethanol to the Washing Buffer

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 kb per preparation.

Content:

• Resuspension Buffer
• Lyticase
  before use, add Lyticase Buffer as indicated on the bottle - store at -20 °C
• Lyticase Buffer
• Lysis Buffer
• RNase A
  before use, add double distilled water as indicated on the bottle - store at -20 °C
• Proteinase K
  before use, add double distilled water as indicated on the bottle - store at -20 °C
• Binding Buffer
• Activation Buffer
• Washing Buffer
  before use, add 96-99 % Ethanol as indicated on the bottle

• Elution Buffer
• Spin Columns and 2 ml Collection Tubes

To be provided by you:
96-99 % Ethanol
Double distilled water
Microtubes 1.5 or 2.0 ml

Preparation procedure:
For S pack (50 preps): Before start, add 500 μl dd-water to the Proteinase K tube, 70 μl Lyticase Buffer to the Lyticase tube, 150 μl dd-water to the RNase A tube and 48 ml 96-99 % Ethanol (not included in the kit) to the Washing Buffer bottle.
For L pack (5 x 50 preps): Before start, add 500 μl dd-water to each Proteinase K tube, 70 μl Lyticase Buffer to each Lyticase tube, 150 μl dd-water to each RNase A tube and 120 ml 96-99 % Ethanol (not included in the kit) to each Washing Buffer bottle.

It is essential to use the correct amount of starting material in order to obtain optimal DNA yield and purify. A maximum amount of
1 - 2 x 10⁷ yeast cells can generally be processed. Overnight cultured yeast 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 8,000 g for 1 min
• Discard the supernatent
• Proceed immediately
• Add 100 μl Resuspension Buffer and 1 μl Lyticase to the cell pellet
• Vortex vigorously for 10 sec
• Incubate for 15 min at 37 °C
• Centrifuge at 10,000 g for 1 min
• Discard supernatant
• Add 300 μl Lysis Buffer and 2 μl RNase A to cell pellet
• Vortex vigorously for 10 sec
• Add 8 μl Proteinase K to the cell lysate and mix by pipetting
• Incubate at 60 °C for 10 min and cool down on ice for 5 min
• Add 300 μl Binding Buffer to the cell lysate
• Vortex briefly
• Place the tube on ice for 5 min
• Centrifuge for 5 min at 10,000 g

B Column Activation [optional]:

• Place a spin column into a 2 ml collection tube
• Add 100 μl Activation Buffer into the Spin Column
• Centrifuge at 10,000 g for 30 sec and immediately proceed to next step
• Discard the flow-through

C Column Loading:

• Pipet the supernatant directly into the spin column
• Centrifuge for 1 min at 10,000 g
• Discard the flow-through

D Primary Washing:

• Add 500 μl Washing Buffer into spin column
• Centrifuge for 30 sec at 10,000 g
• Discard the flow-through

E Secondary Washing:

• Add 500 μl Washing Buffer into the spin column
• Centrifuge for 30 sec at 10,000 g
• Discard the flow-through
• Centrifuge again at 10,000 g for 1 min to remove residual Washing Buffer
• Discard the 2 ml wash tube and place the column in the elution tube

F Elution of DNA:

• Add 40-50 μl Elution Buffer into the center of the column
• Incubate at room temperature for 1 min
• Centrifuge at 10,000 g for 2 min
• Store DNA at 4 °C or -20 °C

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