Purification of DNA from a cell extractIn 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 phaseNucleic acid in aqueous phase

Centrifuge

Remove nucleic acids with pipette

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

Concentration of DNA sampleThe most frequent method of concentration of DNA is ethanol ppt. method

Dilute DNA 1/10 volume of Na-acetate + 3-5 fold of absolute ethanolAt -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

Purification of RNA from a cell extractP19 cells (1.5X106 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

Keep in RT for 2-3 min

Centrifuge at 15000 rpm at 4 C for 20 min

RNA in Aqueous layer Organic 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 % ethanolCentrifuge at 15000 rpm at 4 C for 20 min

Clear RNA pellet

Dried slowly at RT

Dissolve RNA pellet with water or 1X TE

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

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

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