Animal and Fungi DNA Preparation Kit

Description:
Animal and Fungi DNA Preparation Kit is designed for convenient and fast isolation of genomic DNA from animal tissue and fungi. The solution based system minimizes DNA fragmentation that may be problematic in spin-column / filtration based methods. 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.

Content:
Cell Lysis Solution
Cell Resuspension Solution
Proteinase K (before use, solve in double distilled water to obtain a final concentration of 20 mg/ml) – store at -20 °C
Protein Precipitation Solution
Washing Buffer (before use, add 96-99 % Ethanol as indicated on the bottle)
DNA Hydration Solution
RNase A (before use, solve in double distilled water to obtain a final concentration of 4 mg/ml) – store at -20 °C

To be provided by you:
Isopropanol (2-propanol) >99 %
96-99 % Ethanol
Microtubes 1.5 ml

Preparation procedure:
Before start, provide >99 % Isopropanol (2-propanol) (not included in the kit).
For S pack (100 preps): Add 200 μl dd-water to the Proteinase K 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.

1a Cell Lysis for Animal Tissue:

• Transfer 5-10 mg of fresh or frozen tissue to a 1.5 ml microtube.
• Add 300 μl Cell Lysis Solution to the tissue.
• Add 1.5 μl Proteinase K Solution to the lysate and mix by inverting several times.
• Incubate at 55 °C overnight or until tissue has dissolved.

1b Cell Lysis for Fungi:

• Transfer 1 ml of the cultured cells to a 1.5 ml microtube.
• Harvest the cells by centrifuging at 15,000 g for 1 min and discard supernatant.
• Resuspend the cell pellet in 300 μl Cell Resuspension Solution.
• Add 1.5 μl Proteinase K Solution and mix by inverting several times.
• Incubate at 55 °C for 60 min.
• Centrifuging at 15,000 g for 1 min and discard supernatant.
• Resuspend the pellet in 300 μl Cell Lysis Solution.

2 Protein Precipitation:

• Add 100 μl of Protein Precipitation Solution to the cell lysate.
• Mix the solution well by vortexing for 20 sec.
• Centrifuge at 15,000 g for 3 min. (The precipitated protein will be a tight pellet. If the pellet is not tight, repeat mixing, incubate on ice for 10 minutes, and then centrifuge again.)

3 DNA Precipitation:

• Transfer the supernatant to a clean 1.5 ml microtube containing 300 μl of Isopropanol >99 %.
• Mix the sample by inverting gently 50 times.
• Centrifuge at 15,000 g for 1 min. The DNA will be visible as a pellet that ranges in color form off-white to light green.
• Discard the supernatant and drain tube briefly on clean absorbent paper.
• Add 500 μl Washing Buffer and invert 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 to the cell lysate.
• Mix the sample by inverting the tube and incubate at 37 °C for 30-60 min.
• Hydrate the DNA by incubating sample at 65 °C for 60 min.
• Store DNA at 4 °C. For long time storage, place sample at -20 °C or -80 °C.