AIM: To isolate and visualize the genomic DNA from human blood sample by agarose gel

electrophoresis.

PRINCIPLE: Isolation of DNA usually begins with breakdown of cells. Lysed samples are

mixed with phenol, chloroform and isoamyl alcohol for separation of DNA and protein.

Proteins are denatured by the organic mixture. When the sample is centrifuged, DNA is

retained in the aqueous layer, SDS is at the bottom of the tube and denatured proteins form a

cloudy interface. By adding alcohol to the mix, DNA is precipitated. The precipitated DNA is

dissolved in Tris EDTA buffer which can be stored for longer time.

MATERIALS: Polypropylene centrifuge tubes, Eppendorff tubes, Micropipettes, Gloves,

Centrifuge, B1, B2 buffers, Phenol, Chloroform, Isoamyl alcohol, Ethanol. TAE buffer,

Agarose, Electrophoresis Apparatus.

PREPARATION OF REAGENTS:

1. B1 Buffer: Prepare 1000 ml of buffer by adding 1.210g of Tris HCl (10 mM) – pH – 7.6,

0.744g of KCl (10 mM), 2.032g of Mg Cl2 (10 mM) and 0.744g of EDTA (2mM)

2. B2 Buffer: Prepare 1000 ml of buffer by adding 0.121g of Tris HCl (10mM), 0.074g of

KCl (10mM), 0.203g of MgCl2 (10mM), 0.074g of EDTA (2mM), 0.457g of NaCl (0.4mM).

3. Triton-X: Prepare 0.1ml of 100 % triton-x

4. 10% SDS: Prepare 10% of SDS solution by adding 1g of SDS in 10ml of distilled water

5. 6M NaCl: To prepare, add 8.765g of NaCl in 25ml distilled water

PROCEDURE: Collect 1.5ml of citrated blood with the help of syringe into a vial.

2. Add 900 µl of B1 and 50 µl of Triton-X to 300 µl of citrated blood in 1.5 ml vial and incubate

at 370C for 5 minutes.

3. Centrifuge at 8000 rpm for 3 minutes and the discard the supernatant.

4. To the cell pellet, add 300 µl of B2 and 40 µl of 10% SDS.

5. Mix thoroughly and incubate at 370C for 10 minutes, after incubation, add 100 µl of 6M

NaCl and vortex to precipitate the proteins.6. Centrifuge at 8000rpm for 5 minutes and transfer the supernatant into a fresh tube and add

300 µl of isopropanol to it.

7. Centrifuge at 8000 rpm for 10 minutes to pellet down the DNA. Discard the supernatant and

add 70% ethanol and spin at 8000rpm for 5 minutes to remove any excess salts.

8. Discard the supernatant and air dry the pellet, add 30-50 µl of TE buffer to dissolve the

DNA.

9. Prepare 1 % agarose gel and set up the electrophoresis apparatus.

10. Load 20µl of DNA along with tracking dye in the gel and electrophorese at 50-100V. Stain

the gel in ethidium bromide solution and visualize on UV-Transilluminator.