Manual Extraction of DNA from The Blood

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Manual Extraction of DNA from The Blood

• Prepared by Kholoud Al- Homoudi
• Rana Al-Turki

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Materials

• - Blood Sample.
• - Distilled water.
• Dionized water. -
• Ice and Plastic bucket.-

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Equipment

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

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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. -

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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 101
• Phenol.-

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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
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Calculations

• 0.1 ml 100µl
• 1 ml 1000µl
• 100 ml 10000µl

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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.

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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

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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
  10SDS 0.1ml of proteinase K to the pellet.
• Incubate all the tubes over night at 37ºC in
  shaking water bath.

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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.

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Extraction and purification of DNA Procedure
Extraction and purification of DNA Procedure

• Add 3ml of chloroformIsoamyl 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.

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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.

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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.

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Any Question?

• Thank You