DNA Isolation

Description:
The isolation of the DNA involves breaking the cell and the nucleus to
extract the DNA from within, then isolating it from the impurities, proteins,
and other substances.

The isolation of the DNA is done in main

Stages:
Cell dissolution: In this stage the cell and the nucleus are broken to extract
a DNA sample.
Precipitation: In this stage, proteins and impurities are removed from the
sample.
Purification: This stage involves isolating the DNA completely from the
other substances, for a pure DNA sample.

DNA Isolation Procedures:

1. Prepare 10 microliters of Rnase enzyme.

2. Add 700 microliters of dissolution to the sample.

3. Place the sample in the water bath at 37 degrees centigrade for an hour.

4. Add 10 microliters of the Proteinase k enzyme and mix it with the

samples several times with the pipette.

5. Mix the sample well with the mixer for 15 seconds.

(Note: Avoid creating bubbles in the sample.)

6. Place the sample in the water bath at a temperature of 56 degrees

centigrade for two hours.

7. Add 700 microliters of Phenol, Chloroform,and amyl alcohol in the ratios

of 1:24:25 and mix the solution with the sample several times using a
pipette.
8. Place the sample in the centrifuge and set it to 13,000 cycles per minute
for 10 minutes at a temperature of 4 degrees centigrade to isolate the
contents of the tube to two layers.
(Note: To maintain the balance of the system a tube of the same size
filled with water must be added to the opposite side of the sample tube.)

9. Remove the top layer using the pipette, then place it in the clean tube.

10. Repeat steps 7, 8, and 9 twice.

11. Add 700 microliters of Chloroform to the sample you have removed.

12. Place the sample in the centrifuge and set it to 13,000 cycles per minute
for 10 minutes at a temperature of 4 degrees centigrade to isolate the
contents of the tube to two layers.
(Note: To maintain the balance of the system a tube of the same size
filled with water must be added to the opposite side of the sample tube.)

13. Remove the top layer using the pipette, then place it in the clean tube.

14. Repeat steps 12 and 13 once again

15. Add a solution of sodium acetate with a concentration of 3 molar, a tenth

of the size of the sample, to the sample.

16. Add an ethanol solution with a concentration of 95%, double the size of
the sample, to the sample.

17. Mix the content of the tube by turning it. Then leave it to precipitate at a
temperature of - 70 degrees centigrade for a whole day.

18. Place the sample in the centrifuge and set it to 13,000 cycles per minute
for 30 minutes at a temperature of 4 degrees centigrade.
(Note: To maintain the balance of the system a tube of the same size
filled with water must be added to the opposite side of the sample tube.)

19. Pour the floating material in a wastebasket. Retrieve the remaining

material using a pipette.

20. Add 700 microliters of ethanol 70%. Then mix it by pressing it gently to
prevent damage.

21. Place the sample in the centrifuge and set it to 13,000 cycles per minute
for 10 minutes at a temperature of 4 degrees centigrade.
(Note: To maintain the balance of the system a tube of the same size
filled with water must be added to the opposite side of the sample tube.)
22. The liquid is poured into the wastebasket, and the remaining is retrieved
using the pipette gently to preserve the sample and get rid of it.

23. Dry the sample in the air.

24. Dissolve the DNA sample in the solution regulator and mix it with the
sample several times using the pipette.

Note: Now you have a DNA sample and can use it in other operations.