Methods in Anopheles Research - MR4

Chapter 3 : Specific Anopheles Techniques3.5 Protocol for 96 Well DNA ExtractionPage 3 of 411. Centrifuge at 4100rpm for 15 minutes at room temperature.12. Carefully remove and discard supernatant as before.13. Allow remaining ethanol to evaporate from microplate by leaving uncovered at room temperature for30 minutes or until no ethanol remains.14. Resuspend DNA in 50µl of TE buffer to each well.15. Seal microplate with new plastic caps and incubate at 65°C for 1 hour tapping periodically.16. DNA samples can be stored at -20°C or -80°C.Figure 3.5.2 shows the result of 23 An. gambiae s.s., 23 An. arabiensis and 23 desiccated mosquitosamples analyzed with the An. gambiae authentication PCR (Wilkins et al. 2006) and 23 An.quadrimaculatus samples analyzed with the Anopheles ITS2 Amplification (Beebe and Saul 1995), using1µl of template genomic DNA extracted with this protocol.Figure 3.5.1. Example of a 96-bacterial replicator tool usedfor maceration of mosquitosamples and reservoir.Figure 3.5.2. Lanes 1, 50, 51, 100 1kb ladder, lanes 25, 49, 75and 99 control wells. Lanes 2-24 An. gambiae s.s., lanes 26-48 An.quadrimaculatus, lanes 51-74 An. arabiensis and lanes 76-79desiccated mosquito samples. Bands are species specific. 5μl ofsample loaded and run on a 2% agarose EtBr gel.ReferencesBeebe NW, Saul A (1995) Discrimination of all members of the Anopheles punctaulatus complex bypolymerase chain reaction-restriction fragment length polymorphism analysis. The American Journal ofTropical Medicine and Hygiene 53:478-481Collins FH, Mendez MA, Rasmussen MO, Mehaffey PC, Besansky NJ, Finnerty V (1987) A ribosomalRNA gene probe differentiates member species of the Anopheles gambiae complex. American Journal ofTropical Medicine and Hygiene 37:37-41Holelley CE (2007) Economical high-throughput DNA extraction procedure in a 96-well format forDrosophila tissue. Dros Inf Serv 90:137-138Wilkins EE, Howell PI, Benedict MQ (2006) IMP PCR primers detect single nucleotide polymorphisms forAnopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin inAnopheles arabiensis. Malar J 5:125