Yeast DNA Preparation Kit

Description

Yeast DNA Preparation Kit is designed for convenient and fast isolation of genomic DNA from yeast cells. The solution based system minimizes DNA fragmentation that may be problematic in other spin-column/filtration based method.

Because phenol or chloroform is not used it is safe and does not produce any harmful waste. Solution based genomic DNA purification kits guarantee minimal DNA fragmentation and yield DNA sized up to 150 kb.

10 μg purified DNA per preparation can be expected.

For S pack (100 preps):

Add 120 μl Lyticase Suspension Solution to the Lyticase tube, 200 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 (500 preps):

Add 120 μl Lyticase Suspension Solution to each Lyticase tube, 200 μl dd-water to each RNase A tube and 120 ml 96-99 % Ethanol (not included in the kit) to each Washing Buffer bottle.

Buffer — PP-209S
100 preps — PP-209L
500 preps

Cell
Resuspension Solution — 32 ml — 160 ml

Lyticase
(2.5 units/μl) — 300 units — 5x 300 units

Lyticase
Suspension Solution — 130 μl — 650 μl

Cell Lysis
Solution — 32 ml — 160 ml

Protein
Precipitation Solution — 11 ml — 55 ml

Washing Buffer — add 48 ml Ethanol
(final volume 60 ml) — add 120 ml Ethanol to each bottle (final volume 150 ml each)

DNA Hydration Solution — 11 ml — 55 ml

RNase A
(4 mg/ml) — 0.8 mg — 5x 0.8 mg

1 Cell Lysis

• Transfer 1 ml of cultured cells into a 1.5 ml microtube
• Harvest the cells by centrifuging at 15,000 g for 1 min and discard the supernatant
• Resuspend the cell pellet in 300 μl of Cell Resuspension Solution
• Add 1 μl of Lyticase Solution and mix by inverting approx. 25 times
• Place the tube at 37 °C for 30-60 min
• Centrifuge at 15,000 g for 1 min and discard the supernatant
• Resuspend the pellet in 300 μl of Cell Lysis Solution

2 Protein Precipitation

• Add 100 μl of Protein Precipitation Solution and vortex vigorously for 20 sec
• Centrifuge at 15,000 g for 5 min

3 DNA Precipitation

• Pour the supernatant to a clean 1.5 ml microtube containing 300 μl Isopropanol >99 %
• Mix the sample by inverting gently 50 times
• Centrifuge at 15,000 g for 1 min (DNA should be visible as a small white pellet)
• Discard the supernatant and drain tube briefly on clean absorbent paper. Add 500 μl Washing Buffer and invert the tube several times to wash the DNA pellet
• Centrifuge at 15,000 g for 1 min. Discard the ethanol carefully.
• Air dry at room temperature for 10-15 min

4 DNA Hydration

• Add 50-100 μl of DNA Hydration Solution to the dried DNA pellet
• Add 1.5 μl of RNase A Solution and incubate at 37 °C for 30 min
• Hydrate the DNA by incubating for 60 min at 65 °C
• Store the DNA at 4 °C. For long time storage, place sample at -20 °C or -80 °C