Genomic DNA Extraction 

Prepare cell pellet by centrifuging an overnight culture of E. coli at 4°C and 7000rpm,0h 0m 0s for 0h 10m 0s

Gently resuspend the cell pellet in 500µL of autoclaved MilliQ water by mixing with a pipette and transfer into a 2mL microcentrifuge tube (MCT) in a laminar flow hood.

Add 75µL of SDS and 3µL of proteinase K (or how much ever volume is needed to achieve a final concentration of 100 ug/mL).

Heat at 95°C for 0h 5m 0s to inactivate proteinase K.

Let the tube cool to Room temperature

Add 5µL of RNase A (or how much ever volume is needed to achieve a final concentration of 200 ug/mL).

Leave the tube at room temperature for 0h 10m 0s.

Add 1mL of Phenol-Chloroform mixture to the tube.

Centrifuge at 10.000rpm,0h 0m 0s for 0h 10m 0s at 4°C

The contents of the tube should phase separate into three layers: an aqueous layer on top, a viscous jelly-like layer in the middle, and a layer of chloroform at the bottom.

Collect the aqueous layer and the jelly-like layer using a cut tube and transfer them into a fresh MCT.

Add 1mL of Phenol-Chloroform mixture to this tube.

Centrifuge again at 10.000rpm,0h 0m 0s for 0h 10m 0s at 4°C

The contents of the tube should phase separate into three layers as before.

Collect only the aqueous layer at the top and transfer it into a fresh MCT.

Add 160µL of 3Molarity (M) Sodium Acetate to this tube.

Mix gently.

Add 1mL of Isopropanol to the tube and mix gently by inversion till white strands of DNA precipitate out

Centrifuge at 5000rpm,0h 0m 0s for 0h 10m 0s at 4°C

Discard the supernatant.

Add 1mL of Chilled 70% Ethanol gently along the walls of the tube, without disturbing the DNA pellet.

Centrifuge at 5000rpm,0h 0m 0s for 0h 10m 0s at 4°C

Air-dry the tube till there isn't any ethanol remaining.

Resuspend the DNA pellet gently in 100µL of autoclaved MilliQ water by mixing with a pipette under a laminar flow hood.

Store the suspension in a -30°C refrigerator.

Load 1µL of autoclaved MilliQ water as blank on NanoDrop and load 1µL of gDNA suspension as the sample to measure its concentration.

Prepare a 1% agarose gel by adding 0.5g of agarose in 50mL of TAE buffer with 2µL of ethidium bromide.

Load 3µL of 1kb DNA Ladder into the first lane.

Load 1µL gDNA + 1µL dye into the second lane.

Run the gel.

Observe for a single band above the first band of the ladder, close to the well.