Methods in Anopheles Research - MR4

Chapter 3 : Specific Anopheles Techniques3.5 Protocol for 96 Well DNA ExtractionPage 1 of 43.5 Protocol for 96 Well DNA ExtractionClare Holleley and Alice SutcliffeIntroductionThis 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 havetraditionally been conducted by grinding individuals separately in 1.5ml tubes which can be a very timeconsumingprocess, especially when studies require large numbers. Expansion of the salting-out methodto 96-well PCR plates dramatically reduces the amount of time it takes to perform a large number ofextractions. Simultaneous 96-well insect maceration is achieved using a commercially available bacterialcolony replicator tool (Figure 3.5.1). The novel application of this tool as a maceration device coupledwith the salting-out procedure described below radically improves the efficiency of individual genomicDNA extraction without having a large impact on yield or failure. This protocol has been adapted formosquitoes from the original protocol used in Drosophila (Holelley 2007). Using this protocol, we obtainedaverage yields of 6.15µg and 5.65µg per mosquito by processing live and desiccated mosquito samplesrespectively. This compares to yields of 5.90µg and 3.50µg we obtained in our laboratory using the DNAextraction protocol described by Collins et al (1987).MaterialsForcepsIncubator or thermocycler48 or 96-pin bacterial replicator (See Figure 3.5.1)microplates and plastic capsUltra-Centrifuge (capable of holding 96-well microplates)Bunsen burner, striker, and gas sourceReagents1M Tris-HCl (pH 9.0 at 25°C)1M KClTriton® X-1005M Potassium AcetateProteinase K (Roche, 03 115 887)100% Isopropanol70% ethanolTE buffer 0.01 M, pH 7.4Reagent PreparationThermophilic DNA polymerase 1X buffer1. Add 46.95ml deionized water, 2.5 ml 1M KCl, 500µl 1M Tris-HCl (pH 9.0 at 25°C) and 50µl Triton X-100.2. Prepare 5ml aliquots of 1X buffer for long term storage at -20°C otherwise store at 4°C for short termuse.