Chapter 3 : Specific Anopheles Techniques 3.12 96 Well DNA Extraction Protocol v.

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3.12 96 Well DNA Extraction Protocol

Clare Holleley and Alice Sutcliffe Introduction This protocol describes the expansion of a common genomic DNA extraction method (salting-out method) to a 96-well platform adapted for Anopheline mosquitoes. DNA extractions of individual insects have traditionally been conducted by grinding individuals separately in 1.5ml tubes which can be a very timeconsuming process, especially when studies require large numbers. Expansion of the salting-out method to 96-well PCR plates dramatically reduces the amount of time it takes to perform a large number of extractions. Simultaneous 96-well insect maceration is achieved using a commercially available bacterial colony replicator tool (Figure 3.12.1). The novel application of this tool as a maceration device coupled with the salting-out procedure described below radically improves the efficiency of individual genomic DNA extraction without having a large impact on yield or failure. This protocol has been adapted for mosquitoes from the original protocol used in Drosophila (Holelley 2007). Using this protocol, we obtained average yields of 6.15g and 5.65g per mosquito by processing live and desiccated mosquito samples respectively. This compares to yields of 5.90g and 3.50g we obtained in our laboratory using the DNA extraction protocol described by Collins et al (1987). Materials Forceps Incubator or thermocycler 48 or 96-pin bacterial replicator (See Figure 3.12.1) microplates and plastic caps Ultra-Centrifuge (capable of holding 96-well microplates) Bunsen burner, striker, and gas source Reagents 10mM Tris-HCl (pH 9.0 at 25C) 50mM KCl Triton X-100 5M Potassium Acetate Proteinase K (Roche, 03 115 887) 100% Isopropanol 70% ethanol TE buffer 0.01 M, pH 7.4 Reagent Preparation Thermophilic DNA polymerase 1X buffer 1. Add 46.95ml deionized water, 1250 l 1M KCl, 500l 1M Tris-HCl (pH 9.0 at 25C) and 50l Triton X100. This is a 10X solution. 2. Dilute 10X buffer to yield 1X buffer by adding one part 10X buffer to 9 parts sterile water 3. Prepare aliquots of 10X buffer for storage at -20C and store 1X buffer at 4C for short term use.

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Bacterial 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 (5-10 seconds). 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 3.12.1. 2. Aliquot 50l 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 rocking replicator from side to side. It is important that this is not done too vigorously as this can cause the bottom of the well to break or samples to be splashed, contaminating adjacent wells. When extracting DNA from mosquitoes and other insects, it is particularly important to thoroughly macerate the insect to allow cell lysis. 6. Place plastic caps onto microplate and incubate 12-15 hours at 55C. Number of samples 96 1X DNA polymerase buffer Proteinase K 5000l 20l 48 2500l 10l 1 50l 0.2l

Table 3.12.1. Master mix volumes for 96, 48 or one 25l PCR reactions. Amounts for larger master mixes have been adjusted upwards to be for 50 and 100 reactions compensate for imprecise measurements Genomic DNA extraction 1. Remove microplate from thermocycler and cool to room temperature before proceeding. 2. Add 25l 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 60l of 100% isopropanol to each well. 6. Carefully remove the supernatant (approximately 60l) from each well and add to the new microplate containing isopropanol. Avoid dislodging cell pellet. 7. Seal with new plastic caps and invert 30 times to mix. Microplate containing cell pellets can be discarded. 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 well and should not be dislodged or removed. The pellet may appear purple in color. 10. Add 50l of 70% ethanol to each well. Replace caps and invert 20 times to wash DNA. 11. Centrifuge at 4100rpm for 15 minutes at room temperature.

Chapter 3 : Specific Anopheles Techniques 3.12 96 Well DNA Extraction Protocol v. 1 Page 3 of 4 12. Carefully remove and discard supernatant as before. 13. Allow remaining ethanol to evaporate from microplate by leaving uncovered at room temperature for 30 minutes or until no ethanol remains. 14. Resuspend DNA in 50l of TE buffer to each well. 15. Seal microplate with new plastic caps and incubate at 65C for 1 hour tapping periodically. 16. DNA samples can be stored at -20C or -80C. Figure 3.12.2 shows the result of 23 An. gambiae s.s., 23 An. arabiensis and 23 desiccated mosquito samples analyzed with the Anopheles gambiae authentication PCR (Wilkins et al. 2006) and 23 An. quadrimaculatus samples analyzed with the Anopheles ITS2 Amplification (Beebe and Saul 1995), using 1l of template genomic DNA extracted with this protocol.

Figure 3.12.1. Example of a 96-bacterial replicator tool used for maceration of mosquito samples and reservoir. References

Figure 3.12.2. Lanes 1, 50, 51, 100 1kb ladder, lanes 25, 49, 75 and 99 control wells. Lanes 2-24 An. gambiae s.s., lanes 26-48 An. quadrimaculatus, lanes 51-74 An. arabiensis and lanes 76-79 desiccated mosquito samples. Bands are species specific. 5l of sample loaded and run on a 2% agarose EtBr gel.

Beebe NW, Saul A (1995) Discrimination of all members of the Anopheles punctaulatus complex by polymerase chain reaction-restriction fragment length polymorphism analysis. The American Journal of Tropical Medicine and Hygiene 53:478-481 Collins FH, Mendez MA, Rasmussen MO, Mehaffey PC, Besansky NJ, Finnerty V (1987) A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex. American Journal of Tropical Medicine and Hygiene 37:37-41 Holelley CE (2007) Economical high-throughput DNA extraction procedure in a 96-well format for Drosophila tissue. Dros Inf Serv 90:137-138 Wilkins EE, Howell PI, Benedict MQ (2006) IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis. Malar J 5:125

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