1. DNA Extraction Methods Phenol-chloroform DNA extraction from
sand flies 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 ºC for 30
minutes. 3. Incubate The cell lysates with 200ng/ml Proteinase K
at 65 ºC 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 doubl disteled, sterile
water and store it at -20 for PCR experiments. Guanidine
thiocyanate extraction procedure 1. Guanidine thiocyanate
(FLUKA, Switzerland) solution contains 4-M guanidine
thiocyanate, 0.1 M Tris-HCl pH 6.4, 0.02M EDTA pH 8 and 1.3 %
Triton X-100. 2. Sodium iodide (MERCK, Germa