1. Purification of DNA from a cell extract In addition to DNA, bacterial cell wall extract contain significant quantities of protein and RNA. A variety of procedures can be used to remove these contaminants, leaving the DNA in a pure form. Cell extract De-protenize the cell extract Add phenol or 1:1 mixture of Phenol and Chloroform Protein precipitate at the interphase between aqueous phase and organic phase Organic phase Nucleic acid in aqueous phase Centrifuge Remove nucleic acids with pipette

2. Nucleic acids ( DNA and RNA) in aqueous phase Remove RNA specially mRNA by phenol treatment DNA and RNA in aqueous phase DNA in aqueous phase Enzyme ribonuclease or RNAse Precipitate of DNA Centrifuge Dissolve DNA with 1X TE or dH2O

3. Concentration of DNA sample The most frequent method of concentration of DNA is ethanol ppt. method Dilute DNA 1/10 volume of Na-acetate + 3-5 fold of absolute ethanol At -80 C for 30 mins Centrifuge at 4 C for 20 min DNA pellet Wash DNA pellet with 70% ethanol DNA pellet DNA solution for normal storage Centrifuge at 4 C for 10 min Add 1X TE

4. Purification of RNA from a cell extract P19 cells (1.5X10 6 cells) Washed with PBS (Phosphate buffer saline) Cells with PBS Only cells Discard PBS Store at RT for 5 min Add 1 ml TRIzole reagent Centrifuge at 500 rpm at RT Gently vorte for 15 sec. for lysis the cells Add 2 ml CHCl3Shake vigorously for 15 sec Keep in RT for 2-3 min

5. Centrifuge at 15000 rpm at 4 C for 20 min RNA in Aqueous layerOrganic layer 0.5 ml isopropanol Store at RT for 5-10 min Centrifuge at 15000 rpm at 4 C for 20 min RNA pellet Wash RNA pellet with 70 % ethanol Centrifuge at 15000 rpm at 4 C for 20 min Clear RNA pellet Dried slowly at RT Dissolve RNA pellet with water or 1X TE

6. DNA separation by agarose gel electroporesis DNA that have been isolated or purified or DNA that have been generated by digestion with a restriction enzyme can be separated from each other by a simple technique call agarose gel electroporasis Preparation of agar plate 1g agar + 100 ml 1X TE buffer Heat in micro-wave Allow to cool at 55-60 C Poured onto glass plate Keep in RT for solidify the gel for 30 min

7. Process of gel electrophoresis Prepared gel is immersed in 1X TE buffer DNA sample or PCR products are loaded at one end of the gel DNA move through gel by application of an electric current DNA separate according to size

8. Detection of DNA Separated DNA fragments in gel DNA bind with dye and give flurescence under UV radiation DNA bands can be seen and photograph is taken of gel DNA emerged in ethidium bromide dye