Extraction and Quantitation of DNA From E. coli Experiment 6

QUIZ Write the brief procedure for cloning the gene X from the Vibrio fischeri to the E.coli ? Write the open structure of DNA (3-4 bp) by using followings? (no need to draw H bonds) S Pyr Purine P

Why do we isolate DNA ? To perform any experiment with DNA, one should get pure DNA Isolate, analyze, search the genes in different cells, organs, organisms Knock-out the genes and look for their functions Diagnosis of genetic diseases Forensic purposes etc.

Procedure Pour 50 ml of E. coli culture into a 50-ml centrifuge bottle. Centrifuge at 5000 rpm for 5 minutes Discard the supernatant Resuspend the pellet in 6.5 ml of saline-EDTA buffer Pour off the supernatant Add 3.5 ml of saline-EDTA buffer to resuspend the pellet Add 500 µl of SDS Place in a 50 oC water bath for 10 minutes Add 1 ml 5 M NaCl Invert the tube three times to ensure mixing -To get rid of growth medium To inhibit DNAse activity To breakdown membranous structures to provide isotonic medium to denature all proteins to get rid of all fatty membranes To dissociate proteins from DNA

Procedure, continuing Add 4 ml of chloroform-isoamyl alcohol (24:1) Shake vigorously for 15 minutes Centrifuge at 5000 rpm for 3 minutes Carefully decant the top layer (this contains the DNA) into 7.5 ml of cool 95% EtOH Taking a glass rod, carefully come down into the viscous material (DNA plus contaminants), noting the spongy texture and the different colored layers. The top layer is milky white and contains the contaminating proteins of the cell. Holding the glass rod, gently stir the solution. Watch the DNA at the bottom of the beaker winding around the stirring rod. Chloroform causes surface denaturation of proteins, so deproteinizes the DNA solution - Isoamyl alcohol is to reduce foaming, to separate and maintain the stability of the layers of the centrifuged, deproteinized solution. To separate the DNA from the “junk” DNA dissolves in ionic solution, while other macromolecules will not. DNA is precipitated from the ionic solution by ethanol.

Procedure, continuing Absorbance at 260 nm Obtain the materials required: TE Buffer Spectrophotometer (with a UV lamp); quartz or silica cuvettes with a 1 cm light path The extract must be diluted into the range of the standard curve. Dilute the extract with TE according to the table bellow. Label the tubes 1 through 3, place 3 ml of the buffer into tube 4 for a blank.

Procedure, continuing Read the absorbance in a spectrophotometer at 260 nm & 280 nm and calculate the average DNA concentration and purity of your sample. DNA concentration (µg/ml) = (OD 260) x (dilution factor) x (50 µg DNA/ml)/(1 OD260 unit) 1,8 < OD 260 / OD 280 < 2, pure enough OD 260 / OD 280 < 1,8, high protein rate