EFFICIENT AND PRACTICAL APPROACH FOR DNA EXTRACTION




Presenter: IZHAR ALI & BABAR USMAN
Ph.D. ScholarsGUANGXI UNIVERSITYNanning, P.R. China

Purpose of DNA Extraction 

  • To obtain DNA in a relatively purified form which can be used for further investigations, i.e. PCR, sequencing, etc.

    Note: ( i will upload full video after sometime so you may learn from this  blog) and you can download this file in world Click Here



  
     






     Procedure & Protocol

  • Take 1 gm of leaf sample. 
  •  Pulverize plant tissue in liquid nitrogen using a mortar and pestle.
  • Take the grinded material in Eppendorf tubes.
  • Add 800 μl CTAB-Cetyl trimethyl ammonium bromide.
  • Keep the tubes in water bath at 65 °C for 35 minutes and shake three times after each 10 minutes.
  •  After taking sample from water bath, allow the sample to cool at normal temperature.
  •  Add 800 μl of 24:1 CIA (Chloroform Isoamyl Alcohol).
  • Gently shake the sample.
  • Centrifuge the sample at 1300 rpm for 10 minutes.
  • After centrifuge take 550 μl supernatant from the tubes and discard the debris.
  • Again add 550 μl of 24:1 CIA.   
  • Shake the sample gently. 
  • Again centrifuge for 6 minutes at 12000 rpm. 
  • Take supernatant and discard the lower layer. 
  • Add chilled alcohol (Isopropanol) two times than your sample. 
  • Keep the sample in ice box/freezer for 30 minutes.
  • Again centrifuge for 5 minutes.
  •  Discard the liquid material. 
  • Wash the pellet two times with 800 μl of 75% ethanol. 
  • Keep the tube on tissue paper for 30 minutes/over night. 
  • Add 50 μl of ddH2O. 
  • Keep the material in normal temperature for further use.