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℃ 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.

Guanidine thiocyanate extraction procedure

Reagents preparation

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, Germany) is prepared as a 6-M solution.

3. Suspend Three grams of silica beads (Sigma)  in 25 ml of double distilled, sterile water for 24 hours.

4. Centrifuge The suspension  at 12,000 RPM for 5 seconds and remove the supernatant .

5. Add additional 25-ml of double distilled, sterile water  and wash the beads  for 5 hours using rotator.

6. centrifuge The suspension  at 12,000 RPM for 5 seconds and remove the water.

7. Finally, add 30 µl of 1 M of hydrochloric acid (HCl)  and  autoclave the beads

8.   aliquot and store in the dark at room temperature.

Washing buffer

The washing buffer stock contained; 0.2 M Tris HCl pH 7.5, 1 M sodium chloride, and 20 mM EDTA pH 8. 1. Dilute the washing buffer stock solution 1:9 with double, distilled, sterile water

2. Add an equal volume of 100% ethyl alcohol .

3. Divide the solution into 50-ml tubes and store at 20℃.

Procedure

1. ground Each sand fly  in 1.5 ml autoclaved and UV radiated microfuge tubes, using a micro-pestle (Ependorph, Germany).

2. suspend each pestle  in 0.5 ml  4-M guanidine solution and incubate the samples  at 56℃ under gentle agitation.

3. Next day, boil the samples  for 10 minuets and  centrifuge them at 12,000 RPM for 5 minutes

4. Remove the debris  leaving a sand fly tissue pellet.

5. Add 1 ml of 6 M sodium iodide and 10 µl of suspended silica beads  to each tube, vortex gently for 5 seconds and incubate on ice for 1 hour, perform gentle mixing  every 15 minutes.

6. Remove the supernatant carefully and wash the pellet  two times with 500 µl of ice-cold washing buffer.

7. vortex the pellet and centrifuged for 5 seconds at 12,000 RPM.

8. Remove the buffer  and wash the pellet one to two times with 100% ethyl alcohol .

9. Remove ethyl alcohol  and  dry the pellet .

10. Re suspend the DNA   in 100 µl of double distilled, sterile water, mix gently and incubated at 56℃ for 1hour.

11. Centrifuge the DNA samples  at 12,000 RPM for 5 minutes at room temperature,  aliquot the supernatant and store at  20℃ for PCR experiments.