1.Crash Individual sand flies in 0.5 ml lysis buffer (50 mM NaCl ,10 mM EDTA,50 mM Tris-HCl pH 8).2.Incubate The sand fly homogenates with 100 ng/ml Rnase at 37 ℃ for 30 minutes.
3.Incubate The cell lysates with 200ng/ml Proteinase K at 65 ℃ for 2 hours.
4.Extract the DNA with equal volumes of buffered phenol,Phenol-chloroform- isoamyl alcohol (v/v,25:24:1)and finally chloroform-isoamyl alcohol (v/v,24:1).
5.precipitate the DNA with 3-M ammonium acetate and 2.5 volume of 100% ethyl alcohol.
6.wash the DNA pellet with 70% ethanol
7.dry the pellete in speed vacuum centrifuge for 10 minutes.
8.suspend the DNA pellets with 100-µl d oubl disteled,sterile water and store it at -20 for PCR experiments.
