Column-Based DNA Extraction Protocol

Column-Based DNA Extraction with Disruptor Genie

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Pre-Lab Preparation

Materials to Bring

• Lab notebook
• Safety goggles
• Lab coat
• Closed-toe shoes
• Completed pre-lab quiz

Equipment and Reagents

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Protocol

Part 1: Tissue Lysis with Bead-Beating (15 minutes)

Step 1: Sample Preparation

1 Label a ZR BashingBead Lysis Tube with your initials and sample ID

2 Add mosquito tissue to the tube:

• Whole mosquito (1 individual) OR
• Mosquito legs (3-5 legs) OR
• Mosquito thorax (dissected)
• Maximum: 10 mg tissue (larger samples exceed column capacity)

3 Add 750 µL BashingBead Buffer to the tube

4 Cap tube tightly - ensure cap clicks into place

Step 2: Bead-Beating Homogenization

1 Secure tube in Disruptor Genie:

• Place tube in 2 ml tube holder
• Balance with another tube (opposite side)
• Ensure holder is locked in place

2 Set Disruptor Genie to maximum speed

3 Process for 10 minutes:

• Vortexing will be loud - this is normal
• Tissue will be completely disrupted
• Solution will become cloudy/turbid

Step 3: Centrifugation and Clarification

1 Centrifuge the BashingBead Lysis Tube at ≥10,000 x g for 1 minute

• This pellets beads, debris, and insoluble material
• Supernatant contains DNA

2 Transfer up to 400 µL supernatant to Zymo-Spin III-F Filter (in Collection Tube)

• Use P1000 pipette
• Avoid transferring beads or debris
• It's okay if you transfer less than 400 µL

3 Centrifuge at 8,000 x g for 1 minute

• This filters remaining debris
• DNA is in the flow-through (in collection tube)

4 Discard the Zymo-Spin III-F Filter

• Keep the collection tube with filtrate

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Part 2: DNA Binding and Column Purification (10 minutes)

Step 4: Add Binding Buffer

1 Add 1,200 µL Genomic Lysis Buffer to the filtrate from Step 3

• Total volume now: ~1,600 µL
• Mix by pipetting or gentle vortexing

Step 5: Bind DNA to Column (2 rounds)

1 Transfer 800 µL of mixture to Zymo-Spin IC Column (in Collection Tube)

• The column has maximum capacity of 800 µL

2 Centrifuge at 10,000 x g for 1 minute

• DNA binds to silica membrane
• Flow-through contains contaminants

3 Discard flow-through from collection tube

4 Repeat:

• Add remaining ~800 µL mixture to same column
• Centrifuge at 10,000 x g for 1 minute
• Discard flow-through

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Part 3: Washing and Elution (5 minutes)

Step 6: Pre-Wash

1 Transfer column to new Collection Tube

2 Add 200 µL DNA Pre-Wash Buffer to the column

3 Centrifuge at 10,000 x g for 1 minute

4 Discard flow-through

Step 7: Wash

1 Add 500 µL g-DNA Wash Buffer to the column

2 Centrifuge at 10,000 x g for 1 minute

3 Discard flow-through and collection tube

Step 8: Elution

1 Transfer column to clean 1.5 mL microcentrifuge tube

• Label tube with sample ID

2 Add 20-50 µL DNA Elution Buffer directly to column matrix

• Use 20 µL for maximum concentration
• Use 50 µL for maximum total yield
• Recommended: 30 µL (good balance)

3 Incubate 1 minute at room temperature

• This allows DNA to dissociate from silica

4 Centrifuge at 10,000 x g for 30 seconds

• DNA elutes into microcentrifuge tube

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Quality Control

DNA Quantification

Gel Electrophoresis (Optional)

Run 5 µL DNA on 0.8% agarose gel:

• High molecular weight smear: Genomic DNA (10-40 kb)
• Bright band ~16 kb: Mitochondrial DNA
• No low molecular weight smear: Indicates minimal degradation

PCR Compatibility Test

Use 1 µL of extracted DNA as template for COI PCR (as performed in Week 6 PCR lab):

• Successful amplification = high-quality DNA suitable for barcoding

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Troubleshooting

Problem	Possible Cause	Solution
Low yield	Insufficient lysis	Increase bead-beating time to 15 min
Low yield	DNA not binding	Verify Genomic Lysis Buffer added
Low A260/A280	Protein contamination	Ensure complete lysis, repeat wash
Low A260/A230	Salt contamination	Add extra g-DNA Wash Buffer step
No DNA	Elution buffer not added	Double-check elution step
DNA degraded	Over-bead-beating	Reduce time to 5-7 minutes
PCR inhibition	Insufficient washing	Add second wash with g-DNA Wash Buffer

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Comparison with Magnetic Bead Method

Key Differences

Feature	Magnetic Beads	Column
Lysis method	Manual grinding + liquid nitrogen	Bead-beating (Disruptor Genie)
Binding substrate	Magnetic particles	Silica membrane
Separation method	Magnetic rack	Centrifugation
Protocol time	30-40 minutes	15-20 minutes
DNA yield	High (5-10 µg)	Moderate (1-5 µg)
DNA size	High molecular weight (50-150 kb)	Genomic (up to 40 kb)
Best for	Long-read sequencing (Nanopore, PacBio)	PCR, Sanger sequencing, genotyping
Automation	Difficult	Easy (96-well format available)
Cost per sample	Low	Moderate

When to Choose Each Method?

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Real-World Applications

Vector Surveillance Programs

Mosquito control agencies routinely extract DNA from field-collected specimens:

Insecticide Resistance Monitoring

DNA from column extractions is used to genotype resistance alleles:

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Key Takeaways