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, 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.
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 ºC.
1. ground Each sand fly in 1.5 ml autoclaved and UV radiated microfuge tubes, using a micro-pestle
2. suspend each pestle in 0.5 ml 4-M guanidine solution and incubate the samples at 56 ºC under gentle
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 ºC for 1
11. Centrifuge the DNA samples at 12,000 RPM for 5 minutes at room temperature, aliquot the supernatant
and store at –20 ºC for PCR experiments.
2. Polymerase Chain Reaction (PCR )
Taq polymerase: Recombinant Thermus aquaticus DNA polymerase,
PCR buffer: The PCR buffer supplied with the enzyme is 10 times concentrated PCR buffer with ammonium sulfate (NH4)2SO4 which contained (750 mM Tris-HCl PH 8.8 at 25 ºC , 200 mM ammonium sulfate (NH4)2SO4 and 0.1% Tween-20 ).
PCR mix. 50µl reaction volume
1. The PCR mixture contain 10 µl (about 5 ng) genomic DNA, 1x PCR buffer (75 mM Tris-HCl pH 8.8 at 25
ºC, 20 mM ammonium sulfate (NH4)2SO4 and 0.01% Tween-20), 4mM magnesium chloride, 0.2 mM of
deoxynucleotide mixture ( dTTP, dATP, dCTP, dGTP), 1.25 units Taq DNA polymerase and 2µM of
each primer which were previously designed.
2. prepare PCR mixture on ice under hood using UV radiated micropipetes
3. divide the mixture into 0.2 ml thin walled plastic PCR tubes,
4. Add 50 µl of autoclaved mineral oil (Sigma) and transfer to PCR machine (Personal Cycler version 2.71bc,
serial number 9404222, Biotron).
1. 95 ºC for 5 minutes
2. 94 ºC for 3 minutes
3. annealing at 52 ºC for 90 seconds
4. First extension at 72 ºC for 90 seconds
5. Repeat the amplification 44 cycles
6. Final extension time at 72 ºC for 10 minuets.
1. Analyze the PCR product on 3% agarose gel (SeaKem LE agarose, FMC Bioproducts, Rockland, Maine,
USA) using 1X TAE electrophoresis buffer (0.04 M Tris-Acetate, PH 8, 0.001 M EDTA) which stained
with ethidium bromide (200 ng/ml)
2. Run the gel for 30 minuits,
3. Document the gel using a UV camera (Kodak)
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