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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 ؛C for 30 minutes.
3. Incubate The cell lysates with 200ng/ml Proteinase K at 65 ؛C 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.
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