Practical activities N°1

DNA extraction Process

Materials and products required

• Total DNA extracted from fish muscle.

1.Slice 50 mg of muscle sample.

2.Add 400 µl of lysis buffer (10 mM tris-HCl; pH 8.0; 0.4 M NaCl ) and 40 µl of 20% SDS and
10 µl proteinase K 20 mg/ml (sigma-Aldrich), into a sterile 1.5 ml Eppendorf.

3.Mix by vortexing.

4.Incubate the mixture at 65°C for one hour.

5.Add 300 µl of 6 M NaCI to each sample.

6.Vortex the samples at maximum speed for 30 seconds then centrifuge at 10,000 T for 30
minutes at 4°C.

7.Recover the aqueous phase from each sample in a new sterile Eppendorf.

8.Add an equal volume of isopropanol (Sigma-Aldrich), then vortex.

9.Incubate samples at -20°C for one hour.

10.Centrifuge at 10000g for 20 minutes at 4°C.

11.Wash the DNA pellet with cold 7% (v/v) ethanol.

12.Air dry, then re-suspend in 100 µl TE buffer (10mM Tris-HCI, 0.1 mM EDTA , pH 8.0).
 Practical activities N°2

NaCl technical procedure.

Part 01:

1.Thaw 05 or 10 ml of blood at 37°C.

2.Make up to 25 ml in the tube with TE10/10, shake gently and place on ice for 30 minutes.
Centrifuge at 3000 rpm for 15 min.

3.Remove the supernatant, add 15 ml TE10/10, invert the tube to resuspend the pellet, then
complete the tube to the mark.

resuspend the pellet, then complete the tube with 25ml TE10/10.

4.Place the tube on ice for 15 minutes and centrifuge at 3000 rpm for 10 minutes.

5.Repeat this step until a whitish pellet (white blood cell pellet) is obtained.

6.To the lymphocyte pellet, add 1 ml of white cell lysis solution and 25 μl of proteinase K at
20mg / ml proteinase K and homogenise the pellet.

7.Incubate at 37°C over night in a water bath, with gentle agitation.

Part 02:

8.Add 1 ml NaCl, shake vigorously and centrifuge at 4000 rpm for 15 min.

9.Collect the supernatant in another tube, add 2 volumes of cold absolute ethanol and allow the
DNA to precipitate by inverting the tube.

DNA by inverting the tube (formation of the jellyfish).

10.Recover the jellyfish using a sealed Pasteur pipette, rinse it once with 70% ethanol, place it
in an Eppendorf tube and leave it to precipitate and leave to air dry.
11.Dissolve the jellyfish in 100 - 400μl of TE10/1.

12.For complete dissolution, leave the tubes on slow agitation at room temperature for at least
24 hours.

Sodium dodecyl sulphate or SDS is a strongly anionic detergent able to dissolve proteins and
lipids in membranes.

NaCl neutralises the negative charges (carried by the phosphate groups) on the DNA by
removing the H2O molecules surrounding the double helix. This allows it to be precipitated in
alcohol.