Bacteria DNA Preparation Kit - solution-based

Detailní popis

For in vitro use only!

Shipping: shipped at ambient temperature

Storage Conditions: store at ambient temperature

Shelf Life: 12 months

Description:
Bacteria DNA Preparation Kit is designed for convenient and fast isolation of genomic DNA from gram-positive and gram-negative bacteria samples. 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.

Expected yield:
Yields of genomic DNA will vary from sample to sample depending on the amount, quality and type of material processed. An amount of approx. 40 μg purified DNA per preparation can be expected.

Content:
Cell Resuspension Solution
Lysozyme (before use, solve in double distilled water to obtain a final concentration of 100 mg/ml) - store at -20 °C
Cell Lysis Solution
RNase A (before use, solve in double distilled water to obtain a final concentration of 4 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

To be provided by you:
Isopropanol (2-propanol) >99 %
96-99 % Ethanol
Microtubes 1.5 ml
Heating Block or Water Bath at 37 °C and 65 °C

Preparation procedure:
Before start, provide >99 % Isopropanol (2-propanol) (not included in the kit).
For XS pack (20 preps): Add 50 μl dd-water to the Lysozyme tube,
40 μl dd-water to the RNase A tube and 9.6 ml 96-99 % Ethanol (not included in the kit) to the Washing Buffer bottle.
For S pack (100 preps): Add 250 μl dd-water to the Lysozyme 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 250 μl dd-water to each Lysozyme 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.

1a Cell Lysis for Gram-Positive bacteria:

• Transfer 1 ml of cultured cells into a 1.5 ml microtube.
• To harvest the cells centrifuge at 15,000 g for 1 min and discard the supernatant.
• Resuspend the cell pellet in 300 μl of Cell Resuspension Solution.
• Add 2 μl of Lysozyme Solution and mix well by inverting.
• Incubate the tube at 37 °C for 60 min with occasional inverting.
• Centrifuge at 15,000 g for 1 min and discard the supernatant.
• Resuspend the pellet in 300 μl of Cell Lysis Solution.

1b Cell Lysis for Gram-Negative Bacteria:

• Transfer 1 ml of cultured cells into a 1.5 ml microtube.
• To harvest the cells centrifuge at 15,000 g for 1 min and discard the supernatant.
• Resuspend the pellet in 300 μl of Cell Lysis Solution.

2 RNase Treatment:

• Add 1.5 μl of RNase A Solution and mix by inverting.
• Incubate at 37 °C for 15-30 min and cool on ice for 1 min.

3 Protein Precipitation:

• Add 100 μl of Protein Precipitation Solution and vortex vigorously for 20-30 sec.
• Centrifuge at 15,000 g for 5 min.

4 DNA Precipitation:

• Transfer the supernatant to a clean 1.5 ml microtube containing 300 μl Isopropanol >99 %.
• Mix the sample by inverting gently for 1 min.
• 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.

5 DNA Hydration:

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