1. Extraction and Quantitation of DNA From E. coli
Experiment 6

2. 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

4. 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.

5. 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

6. 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.

7. 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.

8. 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