Mosquito DNA Isolation Protocol

This protocol outlines the isolation of high-quality genomic DNA from mosquitoes using a modified CTAB (Cetyl Trimethyl Ammonium Bromide) method.

• Stock Solutions:

  • Phosphate-Buffered Saline (PBS, pH 7.4)
  • Proteinase K Solution (1 mg/mL)
  • TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
  • RNAse A Solution (10 mg/mL) (Optional)
  • CTAB Lysis Buffer (1% CTAB, 0.5 M NaCl, 0.1 M Tris-HCl pH 8.0)

• Working Solutions:

  • Lysis Solution (CTAB Lysis Buffer with Proteinase K)

• Other Materials:

  • Sterile mortar and pestle or bead beating tubes with beads
  • Microcentrifuge tubes (1.5 mL)
  • Micropipettes
  • Pipette tips
  • Microcentrifuge
  • Chloroform:Isoamyl Alcohol (24:1 v/v) (flammable, handle in fume hood)
  • 70% Ethanol (prepared with RNase-free water)
  • RNase-free water
  • Heating block (optional, for proteinase K digestion)
  • DNA quantification instrument (e.g., Nanodrop)
  • Agarose gel electrophoresis equipment (optional, for DNA quality check)

Safety Precautions:

• Wear gloves, lab coat, and safety glasses while handling all materials.
• CTAB is an irritant. Avoid contact with skin and eyes.
• Chloroform is a volatile, toxic solvent. Handle in a fume hood and dispose of according to institutional guidelines.
• Ethanol is flammable. Keep away from open flames.

Protocol:

1. Mosquito Preparation:

   • Freeze mosquitoes at -20°C or store in 70% ethanol at -20°C until use.
   • If using frozen mosquitoes, allow them to thaw completely on ice before proceeding.

2. Homogenization:

   • Transfer a single mosquito (or a pool of few mosquitoes) to a sterile mortar and pestle.
   • Alternatively, add the mosquito(es) to a bead beating tube with sterile beads.
   • Add 200 μL of freshly prepared Lysis Solution (CTAB with Proteinase K).
   • Grind the mosquito thoroughly using the mortar and pestle or homogenize the sample in a bead beater following the manufacturer's instructions.
   • Ensure complete homogenization to lyse the mosquito cells and release the genomic DNA.

3. Proteinase K Digestion:

   • Incubate the homogenate at 55°C for 1-2 hours. Alternatively, incubate at 65°C for 30 minutes if using a heating block for faster digestion.
   • Proteinase K digestion helps degrade proteins that can interfere with DNA isolation.

4. RNAse A Treatment (Optional):

   • Add 10 μL of RNAse A solution (10 mg/mL) to the lysate.
   • Incubate the mixture at 37°C for 30 minutes. RNAse A helps degrade any residual RNA present in the sample. This step can be omitted if RNA contamination is not a concern for your downstream application.

5. DNA Extraction:

   • Add an equal volume (200 μL) of Chloroform:Isoamyl Alcohol (24:1 v/v) to the lysate.
   • Chloroform helps separate the organic and aqueous phases, where DNA partitions to the aqueous phase.
   • Mix the solution thoroughly by vortexing for 30 seconds.

6. Centrifugation:

   • Centrifuge the mixture at high speed (12,000 x g) for 10 minutes at room temperature.
   • Centrifugation separates the mixture into three phases: an upper aqueous phase containing the DNA, a white interphase, and a lower organic phase.

7. Transfer of Aqueous Phase:

   • Carefully transfer the upper aqueous phase (approximately 200 μL) to a clean 1.5 mL microcentrifuge tube without disturbing the interphase or the organic phase. The DNA is present in this aqueous phase.

8. DNA Precipitation:

   • Add an equal volume (200 μL) of ice-cold 70% ethanol to the transferred aqueous phase.
   • Ethanol precipitates the DNA from the solution.
   • Mix the solution gently by inverting the tube several times. Avoid vortexing