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Manual Extraction of DNA from The Blood

Manual Extraction of DNA from The Blood. Prepared by: Kholoud Al- Homoudi Rana Al-Turki . Materials. - Blood Sample. - Distilled water. Dionized water. - Ice and Plastic bucket. -. Equipment. Auto clave. - PH meter. - Balance. -

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Manual Extraction of DNA from The Blood

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  1. Manual Extraction of DNA from The Blood Prepared by: Kholoud Al- Homoudi Rana Al-Turki

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

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

  4. 15ml 50ml Glassware - 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. -

  5. Reagent 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.-

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

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

  8. Extraction and purification of DNA - 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.

  9. Extraction and purification of DNA Procedure 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

  10. Extraction and purification of DNA Procedure Extraction and purification of DNA Procedure 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.

  11. Extraction and purification of DNA Procedure Extraction and purification of DNA Procedure 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.

  12. Extraction and purification of DNA Procedure Extraction and purification of DNA Procedure 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.

  13. Extraction and purification of DNA Procedure Extraction and purification of DNA Procedure 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.

  14. Measure of DNA concentration 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.

  15. Any Question? Thank You

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