ISOLATION OF GENOMIC DNA FROM PLANT TISSUE

 ISOLATION OF GENOMIC DNA

        Isolation of  DNA from the plant tissues is at the heart of      plant molecular biology.   

GENOME :
                 The sum of total of all genetic material of an organism, which stores biological information are called genome.
                 The nature of DNA may be either DNA or RNA.

AIM:
             To isolate DNA from the given sample and quantify the                    DNA.

PRINCIPLE: 
              
             The basic step of DNA isolation are disruption and lysis of cellular structure & separation of the  soluble DNA followed by the removed of protein & outer cell concentration.The concentration of isopropanol will cause the DNA to become insoluble since DNA molecules are ionic.They are highly soluble in water & ethanol are commonly used to precipitate DNA from aqueous solution.

                         
                                

REQUIREMENTS:
              
           - Plant sample (Acalypha indica) ,
           - Extraction Buffer,
           - Centrifuge,
           - Chloroform,
           - Isoamyl alcohol, 
           - Isopropanol,
           - TE Buffer.

EXTRACTION BUFFER PREPARATION :
          
           0.5 M EDTA  PH - 8.0          - 10 ml
           1 M Tris Hcl  PH - 7.4         -  4  ml
           5 M Nacl                               - 28 ml
           Makeup Dis.H20 100 ml    - 58 ml
           0.2 % beta - Mercaptoethanol  - 1  ml


PROCEDURE :
           

•  About 1 g of  fresh leaves was weighed & ground well with 5 ml of extraction buffer and add 2 % w/v CTAB mixed gently (do not stir).

• The sample was given gentle inversions & the homogenate was incubated in a warm water bath maintained at 65*C for 1 hour.  

•  After incubation , the homogenate was brought to room temperature and centrifuged at 10,000 rpm for 10 minutes at room temperature.

• Then , the extract was pipetted out into a sterile 15 ml falcon tube. 

                                    

•    The extract with equal volume of chloroform: Isoamyl               alcohol (24:1) & centrifuged at 8,000 rpm for 10 minutes at       room temperature.

•    After centrifugation the resultant recover the top aqueous           phase was transferred to a new sterile falcon tube and  add         0.7 ml /L volume of isopropanol & mix gently.

•   The sample was given incubation at -20*C for 30 minutes &      then centrifuged at 10,000 rpm for 10 minutes at 4*C.

•    Finally DNA pellet was obtained by decanting the                     supernatant followed by washing with ethanol.

•    The pellet was air dried till the ethanol had evaporated.

•   The air dried pellet was re-suspended  in 0.1 ml of TE                buffer & stored at -20*C for further use.

  Thus the genomic DNA was observed under UV- transilluminator.