DNA Extraction

   EFFICIENT AND PRACTICAL APPROACH FOR DNA EXTRACTION

 

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 some time so you may learn from this  blog)

  

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 emperature.

·      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 icebox/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/overnight.

·      Add 50 μl of ddH2O.

 

Keep the material at normal temperature for further use.