Oct 17, 2021
Genomic DNA Extraction
- 1IISER Pune
- iGEM IISER Pune India 2021
Protocol Citation: Likhith Chandragiri 2021. Genomic DNA Extraction . protocols.io https://dx.doi.org/10.17504/protocols.io.bxeupjew
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: August 17, 2021
Last Modified: October 17, 2021
Protocol Integer ID: 52404
Abstract
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)
Materials
1. Cell Pellet
2. MilliQ Water
3. SDS (10%)
4. Proteinase K (20 mg/mL)
5. RNase A (10 mg/mL)
6. Phenol-Chloroform (1:1)
7. Isopropanol (100%)
8. Chilled 70% Ethanol
9. Sodium Acetate (3M, pH=5.2)
Phenol-Chloroform mixture contains some amount of Isoamyl Alcohol which allows the phase separation to appear distinct
Safety warnings
1. Use gloves while handling Phenol-Chloroform
2. Do not leave Phenol-Chloroform flask open unnecessarily
3. Keep Phenol-Chloroform flask covered completely with aluminium foil, and store in a dark place
Preparing the cell pellet (E coli)
Preparing the cell pellet (E coli)
Prepare cell pellet by centrifuging an overnight culture of E. coli at 4 °C and 7000 rpm for 00:10:00
10m
Extracting the genomic DNA (gDNA)
Extracting the genomic DNA (gDNA)
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.
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).
Heat at 95 °C for 00:05:00 to inactivate proteinase K.
5m
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 00:10:00 .
10m
Add 1 mL of Phenol-Chloroform mixture to the tube.
Centrifuge at 10.000 rpm for 00:10:00 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.
10m
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 00:10:00 at 4 °C
The contents of the tube should phase separate into three layers as before.
10m
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.
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 00:10:00 at 4 °C
10m
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 00:10:00 at 4 °C
10m
Air-dry the tube till there isn't any ethanol remaining.
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.
Using NanoDrop Spectrophotometer to measure gDNA concentration
Using NanoDrop Spectrophotometer to measure gDNA concentration
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.
Verifying gDNA presence using Gel Electrophoresis
Verifying gDNA presence using Gel Electrophoresis
Prepare a 1% agarose gel by adding 0.5 g of agarose in 50 mL of TAE buffer with 2 µL of ethidium bromide.
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.