1. Manual Extraction of DNA from The Blood

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

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

4. - Beakers (50ml, 80ml, 100ml, 500ml). Volumetric flask (100ml, 250ml, 500ml, 1000ml).- - Plastic and glass centrifuge tubes (15ml, 50ml). - Measuring cylinders (50ml, 100ml, 500ml). - Pasteur pipettes. -- Pipettes (1ml, 5ml, 10ml). - Eppendorf tube. - Tips with different size. Racks.- Quarts cuvettes. - 50ml 15ml Glassware

5. Proteinase K (20mg/ml).- Lysis buffer (2X). - SDS 10%. - Salt/ EDTA.- - Chloroform: Isoamyl alcohol. EDTA 0.5M. - Ethanol 99-100%.- 10mM Tris, 1mM EDTA.- 1M Tris PH= 7.6- - TE buffer 10:1 Phenol.- Reagent

6. Number of moles (mole) MolarityVolume (Liters) Weight (Gram) Number of moles Molecular weight (gram/mol) Concentration X Volume = Concentration1 X Volume C X V = C1 X V1 Calculations

7. 0.1 ml = 100µl 1 ml = 1000µl 100 ml = 10000µl Calculations

8. - The DNA was extracted manually from blood sample. - This method uses SDS- proteinase K method which dissolve the the sample and digest the protein component without affecting the DNA. Extraction and purification of DNA

9. Add 5ml of blood + 45ml of Lysis buffer(2X) to 50ml capped centrifuge tube. Mix the samples using the Vortex for 10min. Put the tubes in the Centrifuge for 10min at 3000rpm. There will be 3 layers Extraction and purification of DNA Procedure

10. Discard the supernatant. Add 3ml of EDTA salt buffer + 0.3 ml of 10%SDS + 0.1ml of proteinase K to the pellet. Incubate all the tubes over night at 37ºC in shaking water bath. Extraction and purification of DNA Procedure

11. Add 3ml of liquid phenol to each tube. Mix the samples using the Vortex for 10min. Put the tubes in the Centrifuge for 10min at 2000rpm. There will be tow layers. Extraction and purification of DNA Procedure

12. Add 3ml of chloroform:Isoamyl alcohol to the upper aqueous phase. Put the tubes in the centrifuge for 5 min at 2000rpm. Tow layers will appear. Add 6ml of ethanol to each tube to precipitate the DNA. Extraction and purification of DNA Procedure

13. Put the tubes up side down until the precipitated DNA is completely dry. Add 0.5 ml of 10mM EDTA buffer in 2 ml eppendorf tube to redissolve the DNA over night. Extraction and purification of DNA Procedure

14. 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

15. Thank You