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Manually DNA Extraction Protocol From Blood

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DNA Extraction Protocol to Extract DNA Manually

DNA Can Be Extracted Using These Methods

  • 1. Organic Method (Phenol Chloroform Isoamyl [PCI])
  • 2. Inorganic Method (NaCl 6M)
  • 3. Commercial available kits
  • 4. CTAB (Cetyl trimethylammonium bromide): commonly
    used in the preparation and purification of genomic DNA
    from Plant

Here We Will Discus: Organic Method (Phenol Chloroform Isoamyl [PCI])

 

Material Required

-Lysis buffer (TE)
-Proteinase K
-Phenol-chloroform isoamyl alcohol (PCI)
-SDS 10%
-TNE buffer
-Isopropanol
– Ice cold 95-100% ethanol
– Ultrapure (DNA- & DNase-free) water

 

Required Lab equipment:

-Pippetes,
-1.5mL sterile microcentrifuge tubes or 15, 50 mL Falcons,
-Racks,
-Tips
-Vortex
– Freezer
– Centrifuge

Blood Sample

  • Blood collection in anticoagulant i.e. Ethylenediamide tetra-acetic acid
  • (0.5 M EDTA) containing tube 1.5 mL eppendorf or 15 mL Falcon tube
  • Storage of Blood Samples
  • Field blood samples should be place on ice immediately after their
  • Collection store in freezer at -20°C before DNA extraction

 

Steps in Organic and Inorganic DNA Extraction

  • – Lysis of Red Blood Cells, RBC
  • – Digestion step (Lysis of White blood cells, WBC)
  • – Phase Separation step (Extraction of Protein)
  • – Organic DNA Extraction: PCI
    • Inorganic DNA Extraction: 6M NaCl
  • – DNA Precipitation
  • – Washing with ice cold Ethanol
  • – Dilute the pellet

 

1. Lysis of Red Blood Cells, RBC

Lysis of red blood cells

  • Added 800 uL of Tris EDTA buffer (Tris HCl 10mM, EDTA 2mM) in 200 uL ml of the blood. Mixed by inverting several times.
  • Centrifuged at 5000 rpm for 10 min.
  • Discarded the supernatant.
  • Break the pellet formed at the bottom of the Eppendorf tube by tapping it gently.
  • Add 1 mL TE buffer and mixed it gently.
  • Centrifuged at 5000 rpm for 10 min. this step may be repeated until pallet becomes light pink.

2. Digestion step (Lysis of White blood cells, WBC)

Pellet obtained after lysis of RBCs re-suspended in

– 400 uL Buffer TNE (Tris HCL 10mM, EDTA 2mM, NaCl 400mM),

– 200μL 10% SDS

– 50μL Proteinase K (50μl of 10μg/uL conc.).

Homogenize the tube with gentle rotation Samples incubate at overnight in 58 °C in shaker water bath.


 

Next Day

3. Phase Separation step (Extraction of Protein)

In this step, we can remove the digested protein through

– 6M NaCl (Inorganic Method) or
– Phenol-chloroform isoamyl alcohol (PCI, in ratio 25:24:1 respectively) (Organic Method).
DNA released into solution is extracted with PCI to remove proteinaceous materials.
Add equal volume of phenol-chloroform-isoamyl (PCI) alcohol Mix gently for 2 min and centrifuge for 10 minutes at 10,000 rpm at 4C.

Carefully remove the top (aqueous) phase
containing the DNA using a 1000-ul pipette transfer to a new tube.

 

4. DNA Precipitation

  • Precipitate the DNA with absolute isopropanol and inverted the tubes gently till DNA threads became visible and then left the tubes at room temperature for 10 minutes.
  • Centrifuged at 8000 rpm for 10 minutes and discarded the supernatant carefully and white pellet of DNA may visible at the bottom of the tube.

 

5. Washing with ice cold Ethanol

  • Washed DNA pellet with 1 mL of 70-100% ethanol, break and mix the pellet
  • Then centrifuged at 8000 rpm for 10 minutes and discarded the supernatant carefully
  • Air dried the DNA pellet at room temperature for at least 2 hours

 

6. Dilute the pellet

  • Add 50-100 uL of low T.E. (Tris HCl 10 mL, EDTA 0.2mM) or DEPC water
  • Place the tubes in a shaking water bath at 70°C for one hour so that nucleases were inactivated. Finally DNA will store at –20oC.
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