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Text Box: Leishmania and sand fly Protocols

 

 

Text Box: Molecular Biology

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 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-l doubl 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 l 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 TrisHCl 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 C.

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 C 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 l 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 l 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 l of double distilled, sterile water, mix gently and incubated at 56 C for 1

      hour.

11. Centrifuge the DNA samples  at 12,000 RPM for 5 minutes at room temperature,  aliquot the supernatant 

       and store at 20 C 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 C  , 200 mM  ammonium sulfate (NH4)2SO4  and 0.1% Tween-20 ).

Procedure

PCR mix. 50l reaction volume

 1. The PCR mixture contain 10 l (about 5 ng) genomic DNA, 1x PCR buffer (75 mM Tris-HCl pH 8.8 at 25

     C, 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 2M 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 l of autoclaved mineral oil (Sigma) and transfer to PCR machine (Personal Cycler version 2.71bc,

    serial number 9404222, Biotron).

 

PCR Program

 1.  95 C for 5 minutes

 2. 94 C for 3 minutes

 3.  annealing  at 52 C for 90 seconds

 4. First extension at 72 C for 90 seconds

 5. Repeat the amplification  44 cycles

 6. Final extension time at 72 C for 10 minuets.

 

DNA visualization

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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