Methods in Anopheles Research - MR4

Chapter 3 : Specific Anopheles Techniques3.5 Protocol for 96 Well DNA ExtractionPage 2 of 4Bacterial replicator sterilization:1. Place bacterial replicator in ethanol for 5 minutes ensuring the entire length of the pins is immersed.2. Heat replicator over Bunsen burner or similar until ethanol has evaporated for sterilization (5-10seconds).3. Set bacterial replicator aside to cool to room temperature before use in next steps.OPTIONAL: Other methods for sterilization include autoclaving or 2 hours of UV light.Mosquito preparation:1. Prepare a master mix of buffer as shown in Table 3.5.1.2. Aliquot 50µl of master mix into each well of a 48 or 96-well microplate.3. Using forceps, place one mosquito in each well cleaning forceps between samples.4. Carefully place sterilized, room temperature bacterial replicator into microplate (containing samples).5. Carefully grind mosquito samples for 10 minutes by moving replicator up and down and/or rockingreplicator from side to side. It is important that this is not done too vigorously as this can cause thebottom of the well to break or samples to be splashed, contaminating adjacent wells. When extractingDNA from mosquitoes and other insects, it is particularly important to thoroughly macerate the insectto allow cell lysis.6. Place plastic caps onto microplate and incubate 12-15 hours at 55°C.Number of samples96 48 11X DNA polymerase buffer 5000µl 2500µl 50µlProteinase K 20µl 10µl 0.2µlTable 3.5.1. Master mix volumes for 96, 48 or one 25μl PCR reactions. Amounts for larger master mixeshave been adjusted upwards to be for 50 and 100 reactions compensate for imprecise measurementsGenomic DNA extraction1. Remove microplate from thermocycler and cool to room temperature before proceeding.2. Add 25µl of 5M potassium acetate to each well containing sample.3. Reseal with plastic caps and briefly vortex.4. Centrifuge at 4100rpm and room temperature for 15 minutes to pellet cell debris.5. In a new microplate, add 60µl of 100% isopropanol to each well.6. Carefully remove the supernatant (approximately 60µl) from each well and add to the new microplatecontaining isopropanol. Avoid dislodging cell pellet.7. Seal with new plastic caps and invert 30 times to mix. Microplate containing cell pellets can bediscarded.8. Centrifuge at 4100rpm for 15 minutes at room temperature.9. Carefully remove and discard supernatant. The DNA is should be visible at the bottom of each welland should not be dislodged or removed. The pellet may appear purple in color.10. Add 50µl of 70% ethanol to each well. Replace caps and invert 20 times to wash DNA.