1. Manual Extraction of DNA from The Blood

2. - Blood Sample. - Distilled water. Dionized water. - Ice and Plastic bucket.- Materials

3. Auto clave.- PH meter.- Balance.- - Micro pipette (transfer pipette) (2-20µl, 10-100µl, 100-1000µl) Centrifuge.- Spectrophotometer.- Equipment

4. - Beakers Volumetric flask - - Plastic and glass centrifuge tubes - Measuring cylinders. - Pasteur pipettes. -- Pipettes (1ml, 5ml, 10ml). - Eppendorf tube. - Tips with different size. Racks.- Quarts cuvettes. - 50ml 15ml Glassware

5. 1M Tris PH= 7.6 Lysis buffer (2X). - SDS 10%. - Salt/ EDTA.- EDTA 0.5M. - Ethanol 99-100%.- 10mM Tris, 1mM EDTA.- 1M Tris PH= 8- -TE buffer 10:1 Reagent

6. Add 300µl of blood + 900µl of tris-cl pH = 7.6 (Lysis buffer(2X) ) to an eppendorf tube to lyse red blood cells. Mix the samples and incubate it at room temp. for 1 min Put the tubes in the Centrifuge for 20 sec at maximum speed. There will be 2 layers Extraction and purification of DNA Procedure

7. Discard the supernatant. Add 200 µl of tris-cl pH = 8 + 200 µl EDTA buffer + 200 µl of 10%SDS to the pellet.to lyse WBS Put the tubes in the centrifuge for 5 min at max. speed Extraction and purification of DNA Procedure

8. Take the supernatant in another clean eppendorf Add 60 µl.potassium acetate glecial to precipitate protein Put the tubes in the Centrifuge for 10min at 2000rpm. There will be tow layers. Extraction and purification of DNA Procedure

9. Add 600 µl of ethanol to each tube to precipitate the DNA. Put the tubes in the Centrifuge for 10min at 2000rpm. Discard supernatent Extraction and purification of DNA Procedure

10. Put the tubes up side down until the precipitated DNA is completely dry. Add 100 µl of TE buffer in the eppendorf tube to redissolve the DNA over night. Extraction and purification of DNA Procedure

11. Dilute 25µl of DNA sample with 2ml of distilled water in quartz cuvette and mix throughly. The concentration of DNA sample was assessed by using spectrophotometer. The optical density was recorded at 260 and 280nm. The 260/280nm absorbance ratio was calculated. Measure of DNA concentration

12. Any Question? Thank You