DNA Isolation from spleen

Principle:
Why we choose spleen for DNA isolation:
1. It is a convenient source for the isolation of DNA contains a high quantity of the material.
2. It has low deoxyribonuclease activity which catalyze breking down the DNA into small
fragments..
Reagents:
1. The tissue is homogenized in isotonic saline buffered withj sodium citrate PH7.4:
Because at this ionic strength, the deoxyribonucleoprotein is insoluble and separates well from
other proteins.
2. The sodium citrate or EDTA (Ethylenediaminetetraacetate) is used to:
Inhibit deoxyribonuclease activity by binding Ca++ and Mg++, which are cofactors for this
enzyme.
3. Some DNA isolation protocols used SDS (sodiumdodecylsulfate) to:
Disturb the cell membranes by removing thr lipids and solubilized the protein.
4. Proteinase is used to:
Break down the yield protein during the isolation process.
5. The extraction procedure is carried out under cold condition:
so that any residual DNA'ase activity is minimal.
6. ice-cold ethanol:
The DNA is finally precipitated as a fibrous white mass by the addition of ethanol.
DNA less soluble in cold ethanol.
Note:
The material is best stored frozen and does not undergo any demonstrable change for several
months but drying of the DNA tends to lead to denaturation.

Material:
1.
2.
3.
4.

Spleen
Buffered saline ssc (0.15 mol/l NaCl buffered with 0.015 mol/l sodium citrate,PH7)
Sodium chloride 2M
Ethanol and ether

Methods:

1. Chop 5g of calf spleen into small fragments.

2. Homogenize the spleen with 20ml of buffered saline for 1min.
3. Centrifuge the suspension at 3000rpm for 15 min.
4. Discard the supernatant.
5. Re-homogenize (mix by pipetting) the precipitate in 40ml buffered saline.
6. Centrifuge the suspension at 3000rpm for 15 min.
7. Discard the supernatant.
8. Suspend the combined sediments uniformly in 2mol/l NaCl to a final volume of 100ml.
9. Centrifuge again at 3000rpm for 15 min.
10. Take the supernatant and measure the volume (xml) then transfer it to a clean beaker.
11. Add 2x of the supernatant volume of ice-cold ethanol slowly.
12. Stir the solution continuously with a stirring glass rod while adding the ice-cold ethanol.
13. Spool the fibrous precipitate on a glass rod and leave it to stand in a beaker for 30 min.
14. During this time the clot will shrink and the expressed liquid should be removed by filter
paper.
15. Dissolve the deoxyribonucleoprotein in 100ml of 2mol/l NaCl.
16. Add an equal volume of the chloroform/amyl alcohol mixture (6:1), and blend for 30s.
17. Centrifuge the emulsion at 5000 g for 10-15 min and collect the upper (opalescent)
aqueous layer containing the DNA into a suitable container so that the denatured protein
at the interface of the two liquids is not disturbed.
18. Repeat the treatment with organic solvent twice more and collect the supernatant in a 500
ml beaker.
19. Precipitate the DNA by slowly stirring 2 volumes of ice-cold ethanol with the
supernatant.
20. Collect the mass of fibres on the glass stirring rod.
21. Carefully remove the rod and gently press the fibrous DNA against the side of the beaker
to expel the solvent.
22. Finally, wash the precipitate by dipping the rod into a series of solvents and expelling the
solvent as described above.
a. 70 per cent v/v ethanol
b. 80 per cent v/v ethanol
c. absolute ethanol
d. absolute ether.
23. Let the DNA stand in the fume cupboard for 10min.
24. Weigh the dry DNA.
25. Dissolve the DNA in buffered saline 1 to 10 dilution with distilled water.
26. Store frozen until needed.
Note: this procedure is just for undergrad teaching and it does not have the highest purity for
DNA isolation to be used for researches.