Phlebotomus Sand flies的DNA的提取
1.Crash Individual sand flies in 0.5 ml lysis buffer (50 mM NaCl ,10 mM EDTA,50
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.
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