Genomic DNA Extraction Protocol¶

Using Solarbio Animal Tissue/Cell Genomic DNA Extraction Kit (D1700). This protocol yields high-molecular-weight genomic DNA suitable for PCR, qPCR, Southern blot, restriction digestion, and next-generation sequencing library preparation.

Equipment and Reagents Checklist¶

Item	Recommended / Specification	Notes
Microcentrifuge	12,000–16,000 ×g capability	Pre-cooled if processing RNase-sensitive samples
Heat block or water bath	56°C ± 1°C, 70°C ± 1°C	Preheat before starting
Vortex mixer	Standard	—
Pipettes (10 μL, 100 μL, 1000 μL)	DNase/RNase-free tips with aerosol barrier	Pre-wet tips for viscous solutions
1.5 mL microcentrifuge tubes	DNase/RNase-free	—
96–100% ethanol	Molecular biology grade	Denatured ethanol with additives is NOT acceptable
Sterile forceps / scalpel	For tissue dissection	Clean with 70% ethanol between samples
Liquid nitrogen (optional)	For tissue pulverization	Yields highest-quality DNA from fibrous tissue

Materials — Kit Components per Prep¶

Component	Volume per Prep	Notes
Sample	20 mg tissue or 1×10⁶ cells	See sample-specific notes below
Proteinase K	20 μL	Pre-warmed to RT (viscous solution)
Lysis Buffer LB	200 μL	If precipitate visible, warm to 37°C until dissolved
Binding Buffer BB	200 μL	Must be at RT before use
Wash Buffer WB (ethanol added)	500 μL × 2	Confirm 96–100% ethanol added per label instructions
Elution Buffer EB	50–100 μL	Pre-warm to 56°C for higher recovery

Protocol¶

Step 1: Sample Preparation¶

Time: 5 min.

Tissue: Weigh 20 mg (maximum 30 mg). Cut into pieces < 1 mm³ using a sterile scalpel. Place in a 1.5 mL microcentrifuge tube. For fibrous tissue (heart, muscle, tail), pulverize in liquid nitrogen with mortar and pestle before adding lysis buffer.

Cells: Harvest 1×10⁶ cells (maximum 5×10⁶). Wash twice with 1 mL PBS. Centrifuge at 300×g for 5 min at RT. Discard supernatant completely. For suspension cells, spin directly in the culture tube.

Precaution: Do not exceed the recommended sample input. Excess tissue results in incomplete lysis and low DNA purity.

Step 2: Lysis¶

Component	Tissue (20 mg)	Cells (1×10⁶)
Lysis Buffer LB	200 μL	200 μL
Proteinase K	20 μL	20 μL

Add Lysis Buffer LB and Proteinase K. Vortex thoroughly for 15 s until the sample is fully suspended. For tissue, ensure no large clumps remain.

Incubation: - Tissue: 56°C for 30 min (60 min for fibrous or hard tissue). Vortex for 5 s every 10 min. - Cells: 56°C for 15 min. Vortex once after 7 min.

Expected appearance: The lysate should become clear to slightly translucent. If lysate remains cloudy after 30 min, extend incubation to 60 min and add an additional 10 μL Proteinase K.

Time: 15–60 min.

Step 3: Binding¶

Component	Volume	Instructions
Binding Buffer BB	200 μL	Add, vortex immediately for 5 s
Incubation	70°C, 10 min	Helps denature residual proteins
Ethanol (96–100%)	200 μL	Vortex 10 s immediately after addition

Add Binding Buffer BB and vortex. Incubate at 70°C for 10 min.

Add 200 μL ethanol (96–100%). Vortex immediately for 10 s. The solution should appear homogeneous.

Precaution: If a precipitate forms upon ethanol addition, vortex more vigorously until dissolved. This does not affect DNA binding.

Transfer the entire mixture (≈ 620 μL) to a spin column placed in a collection tube. Centrifuge at 12,000×g for 1 min. Discard the flow-through.

Time: 12 min.

Step 4: Wash¶

Wash Step	Wash Buffer WB	Centrifuge	Discard
Wash 1	500 μL	12,000×g, 1 min	Flow-through
Wash 2	500 μL	12,000×g, 1 min	Flow-through
Dry spin	—	12,000×g, 2 min	Collection tube discarded

Add 500 μL Wash Buffer WB (ensuring ethanol has been added per label instructions). Centrifuge at 12,000×g for 1 min. Discard flow-through.

Repeat the wash step once.

After the second wash, centrifuge at 12,000×g for 2 min to remove residual ethanol. Important: Residual ethanol inhibits downstream PCR and restriction digestion. The dry spin is essential.

Discard the collection tube and place the column in a clean 1.5 mL microcentrifuge tube.

Time: 5 min.

Step 5: Elution¶

Parameter	Standard	For Low DNA	For High Concentration
Elution Buffer EB volume	100 μL	50 μL	30 μL
Incubation	RT, 2 min	RT, 5 min	56°C, 5 min
Centrifuge	12,000×g, 2 min	12,000×g, 2 min	12,000×g, 2 min

Add 50–100 μL Elution Buffer EB to the center of the membrane. Ensure the buffer contacts the membrane directly — avoid pipetting onto the column walls.

Incubate at room temperature for 2 min.

Centrifuge at 12,000×g for 2 min. The eluate contains purified genomic DNA.

For maximum recovery: re-apply the eluate to the column membrane, re-incubate for 2 min, and re-spin.

Time: 4 min.

Quality Checks During Protocol¶

During Lysis¶

Observation	Interpretation
Lysate clear within 15–30 min	Normal; proceed
Lysate remains viscous after 30 min	High molecular weight DNA releasing slowly (normal for spleen, thymus)
Lysate remains cloudy after 30 min	Incomplete protein digestion — extend lysis to 60 min
Clumps visible	Poor homogenization — increase vortexing

After Elution¶

Check	Method	Expected Result
Concentration	UV spectrophotometer (260 nm)	50–350 ng/μL (from 20 mg tissue, 100 μL EB)
Purity A₂₆₀/A₂₈₀	UV spectrophotometer	1.8–2.0
Purity A₂₆₀/A₂₃₀	UV spectrophotometer	≥ 1.8
Integrity	0.7–1% agarose gel	Single band > 20 kb (no smearing)
Functional test	PCR (GAPDH, 35 cycles)	Single band at expected size

Expected Yield¶

Sample	Input	Yield	A₂₆₀/A₂₈₀	Integrity
Mouse liver	20 mg	25–35 μg	1.85–1.95	>30 kb
HeLa cells	1×10⁶	5–10 μg	1.85–1.95	>30 kb
Mouse tail	1 cm (≈20 mg)	10–20 μg	1.80–1.90	15–30 kb
E. coli pellet	1×10⁹	20–40 μg	1.80–1.90	>30 kb
Rat kidney	20 mg	20–30 μg	1.85–1.95	>30 kb
Mouse brain	20 mg	12–20 μg	1.80–1.90	>20 kb

Troubleshooting¶

Issue	Cause	Solution
Low DNA yield	Incomplete lysis	Extend Proteinase K incubation to 60 min; add fresh Proteinase K
	Poor binding	Ensure Binding Buffer BB is at RT; ethanol concentration is 96–100%
	Elution insufficient	Use 50 μL EB pre-warmed to 56°C; incubate 5 min
	Sample over-dried	Do not exceed 2 min dry spin (dried membrane reduces recoverable yield)
A₂₆₀/A₂₈₀ < 1.7	Protein contamination	Add additional Proteinase K; extend lysis time
	Incomplete wash	Ensure Wash Buffer WB has ethanol added; use correct volume
A₂₆₀/A₂₃₀ < 1.5	Chaotrope carryover	Increase wash steps to 3×; ensure dry spin is complete
	Polysaccharide contamination (plant)	Use plant-specific kit D1800
DNA degraded	Nuclease contamination	Use DNase/RNase-free tubes and tips; keep samples on ice before lysis
	Excessive vortexing after lysis	Vortex gently; high molecular weight DNA is shear-sensitive
	Multiple freeze-thaw cycles	Aliquot eluted DNA; do not freeze-thaw > 5×
No DNA in eluate	Ethanol not added to Binding Buffer	Repeat with ethanol; or check Wash Buffer ethanol addition
	Column orientation reversed	Arrow on column must face the same direction as centrifuge rotor hinge
PCR inhibition	Residual ethanol in eluate	Increase dry spin to 3 min; air-dry column for 2 min before elution
	Inhibitor co-purification	Dilute DNA 1:10 in EB or water; re-purify by ethanol precipitation

Optimization Notes¶

Sample Type	Recommended Modifications
Adipose tissue (brain, fat)	After lysis, centrifuge 12,000×g for 5 min at 4°C. Remove fatty top layer before adding Binding Buffer BB.
Hard tissue (bone, cartilage)	Increase Proteinase K to 40 μL; extend lysis to 60 min or overnight at 56°C. Pulverize in liquid nitrogen before lysis.
Fibrotic tissue (scar, heart)	Use 30 mg maximum; increase lysis to 60 min; use bead mill homogenization.
Small tissue samples (< 10 mg)	Reduce all volumes proportionally (100 μL LB, 10 μL PK). Use 30 μL EB for elution.
High cell number (> 5×10⁶)	Split across two columns; or increase LB to 400 μL and PK to 40 μL.
Paraffin-embedded tissue	Use FFPE DNA Extraction Kit (D1850) with overnight deparaffinization and extended Proteinase K digestion.

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