1. DNA Isolation from Haman Blood Cells

2. 2- pellet and supernatant
1- cell extract
2- pellet and supernatant
3- centrifugation
4- rpm and rcf

3. Materials: - Blood with anti-coagulant - Lyses buffer
- Chloroform/ isoamyl alcohol 24:1
- Proteinase K (10mg/ml)
- SE buffer
- SDS reagent (contains EDTA)
- Sodium acetate
- Isopropanol
- Micropipettes and tips
- Refrigerated centrifuge and
-15 ml Eppendorf tube

4. Steps:
1- In Eppendorf tube add 2.5 ml of blood with 7.5 ml of lysis buffer, shake well, and leave it in ice for 5 min.
2- Put the sample in the centrifuge (with balancing tube of 10 ml water) at 1200 rpm, 4˚C for 10 min.
3- Discard supernatant. Add to the pellet 2.5ml of lyses buffer and centrifuge again at same conditions.
4- Discard supernatant. Add 1.25 ml SE buffer to pellet, centrifuge again at same conditions.
5- Discard supernatant. Add 1.25 ml of SE buffer + 10μl proteinase K + 60 μl SDS and shake gently for 7 min.

5. 6- Add 1. 25 ml SE buffer + 2. 5 ml phenol
6- Add 1.25 ml SE buffer ml phenol. Shake well for 5 min then centrifuge again at 3000 rpm, 10 ˚C for 5 min.
7- Transfer supernatant to new tube. Add 2.5 ml Phenol/ chloroform/isoamy alcohol. Shake well for 5 min, then centrifuge again at same conditions.
8- Transfer supernatant to new tube. Add 2.5 ml chloroform/isoamyl alcohol. Shake well for 5 min, then centrifuge again at same conditions.
9- Transfer supernatant to new tube. Add 75 μl 3M Sodium acetate ml isopropanol. Observe DNA fibers visually.

7. Isolation techniques for Cell Components

8. Isolation of Glycogen Granules
Glycogen is a colorless solid material that can be dissolved in cold water to form a viscous solution . Glycogen gives red color with iodine.

11. Material:
- Rat that has been fed with starchy food
- Mortar and pestle apparatus
- 10 % trichloroacetic acid
- Centrifuge and centrifuge tubes
- Alcohol and iodine.

12. steps:
1- Dissect rat and take its liver, cut it to small pieces. In a mortar crush liver with grated ice and cooled washed water with cold trichloroacetic acid (10%) (1 ml of acid for each 1 gram of liver).
2- Put mixture in glass beaker, then distribute it into centrifuge tubes.
3- Put centrifuge tubes into the centrifuge at 2000 rpm for 5 min.
4- Take the upper supernatant containing glycogen into a 200 ml flask, and discard the pellet.
(To insure obtaining maximum amount of glycogen , wash the mortar with cold solution of trichloroacetic acid and centrifuge again).

13. 5- Take the upper supernatant into flask , and discard pellet.
6- Add 95% alcohol to the supernatant (where amount of alcohol is twice amount of liver extract), shake flask well and leave it aside. Observe formation of precipitant. We can accelerate formation of precipitant by adding salt and warming the mixture.
7- Discard supernatant, and collect the white precipitant (glycogen). We can further purify the glycogen by rewash it with water and precipitate with alcohol.
8- Apply small amount of glycogen into a clean glass slide, then add a drop of dil. iodine solution. Examine slide in microscope.

14. (2) Isolation of Mitochondria from Liver

15. In the experiment, liver extract will be centrifuged 3 times:
To study cell’s organelles and other components, cytoplasmic membrane should be destroyed (lysed) with conserving components structure. The result from lysis is called cell extract.
In the experiment, liver extract will be centrifuged 3 times:
First : nuclei and other heavy cellular parts will precipitate, and mitochondria will be in the supernatant.
Second: Mitochondria precipitate in pellet.
Third: Mitochondria separate from other components as proteins, and mitochondria will precipitate in pellet as final result.

16. Steps:
1- Cut a 2 gm piece of liver, put it in mortar with 18 ml of homogenizing buffer
2- Grind mixture with electric homogenizer for 2 minutes
3- Put 5 ml of homologous mixture in 15 ml centrifuge tube
4- Put centrifuge tubes in centrifuge centrifuge at 2400 rpm for 10 min.
5- Transfer supernatant in new centrifuge tube

17. 6- Centrifuge for maximum speed (6000 rpm) for 10 min
7- Discard supernatant, and add 5 ml of suspension buffer on pellet, and mix with dropper to make homologous mixture
8- Centrifuge at maximum speed for 10 min.
9- Discard supernatant, add 40 ml of suspension buffer
10 – In new test tube mix 6 drops of Jenus green B stain μl of mitochondria suspension , and observe color of suspension.
11- Apply a drop of suspension on a slide, cover with cover slide and examine with microscope high power.