Mammalian Genomic DNA Extraction Kit (Proteinase K Method) Protocol

Mammalian Genomic DNA Extraction Kit (Proteinase K Method) is a complete, optimized reagent system designed to efficiently isolate high-molecular-weight genomic DNA from a broad range of mammalian tissues and cultured cells using Proteinase K-mediated lysis followed by column-based purification.

SKU

M1527094

Method

Proteinase K digestion followed by spin-column purification

Sample Types

Mammalian tissues and cultured cells

Key Output

High-molecular-weight, high-purity genomic DNA suitable for PCR, Southern blotting, and next-generation sequencing

Reference Catalog

Leagene NE0201

Principle and Overview

The Mammalian Genomic DNA Extraction Kit (Proteinase K Method) relies on a two-stage process. In the first stage, Proteinase K — a broad-spectrum serine protease — is used under denaturing buffer conditions to digest proteins, including histones and other DNA-binding proteins, releasing genomic DNA from the nuclear matrix. Detergent-based lysis buffers simultaneously disrupt cell and nuclear membranes, ensuring complete release of nucleic acids.

In the second stage, the lysate is applied to a silica membrane spin column. Genomic DNA binds selectively to the membrane under high-salt chaotropic conditions, while proteins, lipids, and other contaminants are removed by successive wash steps. Pure, high-molecular-weight genomic DNA is finally eluted with a low-salt elution buffer or nuclease-free water, ready for downstream molecular biology applications.

Materials and Reagents

Kit components (refer to the kit insert for exact volumes and storage conditions):

• Cell/Tissue Lysis Buffer
• Proteinase K solution
• RNase A solution (optional, for RNA removal)
• Binding Buffer (chaotropic, high-salt)
• Wash Buffer I
• Wash Buffer II (ethanol-based)
• Elution Buffer or nuclease-free water
• Silica membrane spin columns with collection tubes

Additional materials required (not supplied):

• Microcentrifuge capable of ≥13,000 × g
• Heating block or water bath (56 °C and 70 °C settings)
• Pipettes and sterile, nuclease-free tips
• 1.5 mL and 2 mL microcentrifuge tubes
• 100% ethanol (molecular biology grade)
• Phosphate-buffered saline (PBS), ice-cold

Note: Exact component names and volumes may vary; always consult the current kit insert supplied with SKU M1527094.

Step-by-Step Protocol

Step 1 — Sample Preparation

For cultured cells: collect cells by centrifugation (300 × g, 5 min), aspirate medium, and wash the pellet once with ice-cold PBS. For tissue: excise fresh or frozen tissue (typically ≤25 mg), mince finely on ice with a sterile blade, and transfer to a 1.5 mL tube.

Step 2 — Lysis and Proteinase K Digestion

Add the appropriate volume of Lysis Buffer and Proteinase K solution to the sample. Mix thoroughly by vortexing or pipetting, then incubate at 56 °C for 1–3 hours (or until complete lysis is achieved), with occasional mixing. If RNA removal is desired, add RNase A and incubate at room temperature for 5 minutes before proceeding.

Step 3 — Binding

Add the specified volume of Binding Buffer to the lysate and mix well. Transfer the mixture to a silica membrane spin column seated in a 2 mL collection tube. Centrifuge at ≥10,000 × g for 1 minute. Discard the flow-through.

Step 4 — Washing

Apply Wash Buffer I to the column and centrifuge at ≥10,000 × g for 1 minute; discard flow-through. Repeat with Wash Buffer II. Perform a final dry spin at ≥13,000 × g for 2 minutes to remove residual ethanol, which can inhibit downstream enzymatic reactions.

Step 5 — Elution

Transfer the spin column to a clean 1.5 mL microcentrifuge tube. Apply pre-warmed Elution Buffer (70 °C) or nuclease-free water to the membrane center, incubate at room temperature for 2–5 minutes to improve yield, then centrifuge at ≥10,000 × g for 1 minute. Collect the eluate containing purified genomic DNA. For maximum yield, a second elution with a smaller volume may be performed.

Step 6 — Quality Assessment

Measure DNA concentration and purity by spectrophotometry (A260/A280 ratio of 1.7–1.9 indicates high purity). Optionally, run an aliquot on a 0.8% agarose gel to confirm high-molecular-weight integrity. Store purified DNA at −20 °C for long-term storage or at 4 °C for short-term use.

Troubleshooting

Problem	Possible Cause	Recommended Solution
Low DNA yield	Incomplete lysis; insufficient Proteinase K digestion time	Extend incubation at 56 °C; ensure proper mixing throughout digestion
Low A260/A280 ratio (<1.7)	Protein contamination; insufficient washing	Repeat wash steps; ensure Wash Buffer II ethanol content is correct
Low A260/A230 ratio (<1.8)	Residual chaotropic salts or ethanol	Add an extra Wash Buffer II step; perform an additional dry spin before elution
DNA degradation (smearing on gel)	Nuclease contamination; improper sample handling	Use nuclease-free reagents and tubes; process samples on ice; store DNA at −20 °C
Column clogging	Excess starting material; incomplete lysis	Reduce sample input; extend lysis time; centrifuge lysate before loading to column

Frequently asked questions

What types of mammalian samples are compatible with this kit?

The kit is designed for a broad range of mammalian sources, including fresh, frozen, or cultured cells, as well as soft and fibrous tissues. Samples such as blood, spleen, liver, muscle, and tail clips from rodents are commonly processed. Very fibrous or high-fat tissues may require additional homogenization or extended lysis times.

How should I assess the purity and integrity of the extracted genomic DNA?

Purity is best assessed by UV spectrophotometry: an A260/A280 ratio of 1.7–1.9 indicates protein-free DNA, while an A260/A230 ratio above 1.8 indicates low salt or solvent contamination. DNA integrity can be confirmed by running an aliquot on a 0.8% agarose gel, where high-quality genomic DNA appears as a tight, high-molecular-weight band with minimal smearing.

Can the extracted DNA be used directly for PCR and next-generation sequencing?

Yes. When extracted according to this protocol, the purified genomic DNA is compatible with standard downstream applications including PCR, quantitative PCR (qPCR), Southern blotting, restriction enzyme digestion, and next-generation sequencing library preparation. Ensure the final dry-spin step is completed to remove residual ethanol, which can inhibit enzymatic reactions.

How should the extracted genomic DNA be stored?

For short-term use (up to one week), store purified genomic DNA at 4 °C in Elution Buffer or nuclease-free water. For long-term storage, aliquot and store at −20 °C to avoid repeated freeze-thaw cycles, which can degrade high-molecular-weight DNA. Avoid storing DNA in plain water at room temperature, as this promotes hydrolysis and nuclease activity.

References

1. Leagene Biotechnology. Mammalian Genomic DNA Extraction Kit (Proteinase K Method), Product No. NE0201, Kit Insert/Instruction Manual.
2. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, 2001.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.