TITLE: Genomic DNA Extraction 
AUTHORS: Likhithchandragiri  

[DESCRIPTION]
Genomic DNA extraction from E. coli

[GUIDELINES]
1. Don't place any vials containing SDS into an ice tray as the SDS will precipitate 
2. Use a laminar flow hood while working with any cultures in autoclaved reagents or cultures
3. Don't vortex any tubes  
4. Dab with a paper towel before putting any new sample on NanoDrop 
5. Look for a peak at 260 nm, and 260/280 and 260/230 ratios over ~1.8 to confirm a pure gDNA extract
6. NanoDrop may provide an overestimate of DNA concentration (due to the presence of other contaminants like RNA)

[STEPS]
SECTION: Preparing the cell pellet (E coli)
1. Prepare cell pellet by centrifuging an overnight culture of E. coli at 4 °C and  7000 rpm  for 10 min  

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

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


SECTION: Extracting the genomic DNA (gDNA)
4. Heat at 95 °C for 5 min to inactivate proteinase K.

SECTION: Extracting the genomic DNA (gDNA)
5. Let the tube cool to Room temperature 

Add 5 µL  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 10 min.

SECTION: Extracting the genomic DNA (gDNA)
6. Add 1 mL  of Phenol-Chloroform mixture to the tube.

Centrifuge at 10.000 rpm for 10 min 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.
SECTION: Extracting the genomic DNA (gDNA)
7. Collect the aqueous layer and the jelly-like layer using a cut tube and transfer them into a fresh MCT.

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

Centrifuge again at 10.000 rpm  for 10 min at 4 °C 

The contents of the tube should phase separate into three layers as before.
SECTION: Extracting the genomic DNA (gDNA)
8. Collect only the aqueous layer at the top and transfer it into a fresh MCT.

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

Mix gently.
SECTION: Extracting the genomic DNA (gDNA)
9. Add 1 mL  of Isopropanol to the tube and mix gently by inversion till white strands of DNA precipitate out 

Centrifuge at 5000 rpm for 10 min at 4 °C 

SECTION: Extracting the genomic DNA (gDNA)
10. Discard the supernatant.

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

Centrifuge at 5000 rpm for 10 min at 4 °C 

SECTION: Extracting the genomic DNA (gDNA)
11. Air-dry the tube till there isn't any ethanol remaining.


SECTION: Extracting the genomic DNA (gDNA)
12. Resuspend the DNA pellet gently in 100 µL of autoclaved MilliQ water by mixing with a pipette under a laminar flow hood.

Store the suspension in a -30 °C  refrigerator.

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



SECTION: Verifying gDNA presence using Gel Electrophoresis
14. Prepare a 1% agarose gel by adding 0.5 g of agarose in 50 mL of TAE buffer with 2 µL of ethidium bromide.

SECTION: Verifying gDNA presence using Gel Electrophoresis
15. Load 3 µL of 1kb DNA Ladder into the first lane.

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

Run the gel.

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