ENTM201L - General Entomology Laboratory | UC Riverside
This protocol uses magnetic beads to extract high molecular weight genomic DNA from mosquito specimens. The magnetic bead method is ideal for long-read sequencing applications and produces DNA fragments ranging from 50-150 kb.
1 Label a 1.5 mL microcentrifuge tube with sample ID
2 Record sample information in lab notebook
1 If preserved in ethanol, air dry for 5 minutes
2 Place single mosquito in labeled tube
3 Add 20 µL of Proteinase K to the tube
1 Add 200 µL Lysis Buffer (LB)
2 Use clean pestle to grind tissue thoroughly
3 Ensure complete homogenization (no visible tissue pieces)
Thorough grinding is critical for high DNA yield. Grind until no visible tissue pieces remain.
1 Vortex for 15 seconds
2 Incubate at 56°C for 30 minutes
3 Vortex briefly every 10 minutes during incubation
Proteinase K digests proteins at 56°C, releasing DNA from cellular structures. Periodic vortexing ensures complete tissue breakdown.
1 Vortex magnetic bead stock solution for 30 seconds
2 Add 20 µL magnetic beads to the lysate
3 Add 200 µL Binding Buffer (BB)
Always vortex magnetic beads immediately before use. Beads settle quickly and must be evenly suspended.
1 Vortex thoroughly for 10 seconds
2 Incubate at room temperature for 10 minutes
3 Mix by inverting tube every 2 minutes
The Binding Buffer creates conditions for DNA to adhere to the magnetic bead surface. Periodic mixing ensures all DNA molecules contact the beads.
1 Place tube on magnetic rack for 2 minutes
2 Wait until solution clears and beads collect at magnet
3 Carefully pipette off and discard supernatant
DO NOT disturb the bead pellet when removing supernatant. Keep tube on magnetic rack and pipette from the side opposite the magnet.
1 Remove tube from magnetic rack
2 Add 500 µL Wash Buffer 1 (W1)
3 Vortex for 5 seconds to resuspend beads
4 Place on magnetic rack for 1 minute
5 Remove and discard supernatant
Wash Buffer 1 removes proteins and cellular debris while DNA remains bound to magnetic beads.
1 Add 500 µL Wash Buffer 2 (W2)
2 Vortex for 5 seconds
3 Place on magnetic rack for 1 minute
4 Remove and discard supernatant
1 Repeat step 9 with fresh Wash Buffer 2
2 After removing supernatant, briefly spin tube
3 Place back on magnetic rack
4 Remove any residual wash buffer with P20 pipette
Removing all wash buffer is critical. Residual ethanol will inhibit downstream applications. Use a P20 pipette to remove the last few microliters.
1 Keep tube on magnetic rack
2 Air dry beads for 5 minutes (cap open)
3 Beads should appear matte, not shiny
Do not over-dry beads (>10 minutes). Over-dried beads are difficult to resuspend and reduce DNA recovery.
1 Remove tube from magnetic rack
2 Add 50 µL Elution Buffer (pre-warmed to 65°C)
3 Vortex vigorously for 10 seconds
4 Incubate at 65°C for 5 minutes
Warm, low-salt Elution Buffer releases DNA from the magnetic beads. Elevated temperature improves DNA recovery.
1 Place tube on magnetic rack for 2 minutes
2 Transfer clear supernatant (contains DNA) to new labeled tube
3 This is your purified DNA sample
When transferring eluted DNA, avoid aspirating any magnetic beads. Leave 1-2 µL behind if needed to prevent bead contamination.
1 Measure concentration using NanoDrop or Qubit
2 Record A260/A280 and A260/A230 ratios
3 Expected yield: 1.5-2 ng/µL (typical for mosquito specimens)
1 Store at -20°C for short term (weeks)
2 Store at -80°C for long term (months/years)
3 Label clearly with date, sample ID, and concentration
| Parameter | Expected Value |
|---|---|
| Concentration | 1.5-2 ng/µL |
| Total yield | 75-100 ng DNA (in 50 µL elution) |
| A260/A280 ratio | 1.8-2.0 (pure DNA) |
| A260/A230 ratio | 2.0-2.2 (no contaminants) |
| Parameter | Magnetic Beads | Spin Columns |
|---|---|---|
| Time | ~2 hours | ~1.5 hours |
| Yield | Higher | Moderate |
| Purity | Excellent | Good |
| Hands-on time | Less | More |
| Automation | Possible | Limited |
| Cost per sample | Higher | Lower |