Genomic DNA Purification using illustra™ GenomicPrep Mini Spin Kits

illustra™ genomicPrep Mini Spin Kits from Cytiva are designed for the rapid extraction and purification of genomic DNA. The protocols have been designed to minimize shearing, resulting in high-quality genomic DNA that is compatible with most molecular biology techniques, including cloning, restriction enzyme digestion, PCR amplification, and genotyping applications.

The developed method uses detergents and a chaotropic agent to extract DNA, denature protein components, and promote the selective binding of DNA to the silica membrane contained in an illustra™ mini column (5, 6). Proteinase K is the protease of choice to digest protein from samples, because it is active even when enzyme inhibitors such as EDTA and detergents are present in samples. Denatured contaminants are easily removed by subsequent washing of the silica membrane with an ethanolic buffer. The purified genomic DNA is eluted in a low-ionic-strength buffer at a concentration suitable for most downstream molecular biology applications. Purity is high—A₂₆₀/A₂₈₀ readings are usually 1.7 to 1.8.

illustra™ tissue & cells genomicPrep Mini Spin Kit has been optimized to extract DNA from several tissue types such as liver, kidney, and mouse tails. Typical yields are 0.5 to 1.5 μg of genomic DNA per mg of tissue. Between 5 and 50 mg of tissue can be used per miniprep. Purified genomic DNA can be obtained in about 90 min.

illustra™ blood genomicPrep Mini Spin Kit is designed for use with whole blood, buffy coat, bone marrow, and nucleated RBC and can process in the range of 50 to 1000 µL of whole blood. Purified genomic DNA yields are typically between 4 and 6 μg from 200 µL of whole blood. Purified genomic DNA can be obtained in as little as 20 min.

illustra™ bacteria genomicPrep Mini Spin Kit has been optimized to extract genomic DNA from Gram-negative and Gram-positive bacteria with yields ranging from 4 to 12 μg of genomic DNA per purification. Bacterial numbers ranging from 1 to 4 × 10⁹ cells in 1 mL can be used for each purification. Purified genomic DNA can be obtained in as little as 40 min.

A general protocol for genomic DNA purification using illustra™ genomicPrep Mini Spin Kits is provided below.

Materials

illustra™ mini columns, buffers, lyophilized Proteinase K, and collection tubes are provided with each kit

Water bath or heating block for 70 °C incubation

RNase A (optional): 20 mg/mL

Dulbecco’s PBS solution is required for tissue homogenization or cell resuspension and for blood samples < 200 µL

Homogenizer for animal tissue, for example, hand-held motor-driven homogenizer (Kimble, part no. 749540 or equivalent) with probe (part no. 749520-0090).

For blood samples > 300 µL

RBC lysis buffer is required. See Advance Preparation, below.

For Gram-positive bacteria only

Lysozyme buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.1 M NaCl, 5% Triton™ X-100) Lysozyme (10 mg/mL in 10 mM Tris-HCl, pH 8.0)

Advance preparation

Dissolve Proteinase K in water.

Add ethanol to wash buffer.

Heat elution buffer.

For tissue samples: homogenize thoroughly.

RBC lysis buffer

This buffer is needed only for processing blood and/or blood fractions ranging from 300 to 1000 µl using the two-stage lysis method as described in the protocol for this volume range.

See product instruction for Blood Mini Spin Kit for details.

10 mM KHCO₃

155 mM NH₄Cl

0.1 mM EDTA, pH 8

Filter sterilize using a 0.2 µm filter. The volume of RBC Lysis buffer required per purification is three times the volume of blood to be processed.

PBS

Dissolve the following in 800 mL of distilled H₂O:

8 g of NaCl

0.2 g of KCl

1.44 g of Na₂HPO₄

0.24 g of KH₂PO₄

Adjust pH to 7.4.

Adjust volume to 1 liter with additional distilled H₂O.

Sterilize by autoclaving.

Alternatively, PBS can be purchased as a ready-made solution or as tablets for adding to water prior to autoclaving.

Protocol

Note: Columns and buffers are NOT transferable between Cytiva kits in the illustra™ product range. Please ensure you use the correct columns and buffers for your purification.

1. Prepare sample

a. Homogenize animal tissue

Homogenize 5 to 50 mg of tissue in a small volume of PBS.

b. Resuspend mammalian cells

Resuspend cells in a small volume of PBS.

c. Harvest bacterial culture

Pellet bacterial cells (1 mL) and remove culture medium.

d. Prepare blood

For 50 to 300 µL of blood

For blood samples < 200 µL, dilute up to 200 µL with PBS. Proceed to step 2, lysis. For 300 to 1000 µL of blood: We recommend a two-stage lysis of blood and its fractions. The first stage involves selective lysis of RBC, while WBC are pelleted and resuspended to 200 µL. In the second stage, nucleated cells are lysed to release DNA that is then purified from the silica membrane column. See product instructions for the Blood Mini Spin Kit for details.

2. Lyse cells

a. Animal tissue, cultured cells, and 50 to 300 µL of blood

Add detergent-containing lysis buffer and Proteinase K. Chaotropic salts in the lysis buffer break open the cells, and Proteinase K digests proteins. Incubate briefly at room temperature (for blood) or for 1 h with heat (for tissue and cells).

b. Gram-negative bacteria

Add two lysis buffers and Proteinase K in the correct sequence. Lysis buffers break open the cells via detergents and osmotic shock, and Proteinase K digests proteins.

c. Gram-positive bacteria

Add lysozyme buffer and lysozyme. After incubation, add Proteinase K.

3. Remove RNA (optional)

Add RNase and incubate at room temperature. RNase treatment is strongly recommended for cultured mammalian cells as most laboratory-grown cells contain large quantities of RNA.

4. Bind genomic DNA

The chaotropic salt in the lysis buffer promotes selective binding of genomic DNA to the silica membrane. Denatured proteins are collected in the flowthrough following centrifugation. Do not overload the column. The maximum volume that can be loaded is 720 µL.

5. Wash and dry

Wash the mini column with lysis buffer, followed by wash buffer to remove chaotropic salts and other contaminants.

6. Elute purified genomic DNA

Elute genomic DNA in a low-ionic-strength buffer containing EDTA or simply water if downstream applications are sensitive to EDTA. For short-term storage, place genomic DNA at 4 °C. For long-term storage, aliquot sample and store at -20 °C. Do not subject samples to repeated freeze-thaw cycles.