Chromosomal DNA isolation

Obtaining high-quality and intact DNA is a critical first step for a wide range of molecular biology experiments. DNA extraction and purification techniques isolate DNA from its cellular milieu, removing cellular debris, proteins, and other contaminants that can interfere with downstream applications.

The choice of specific DNA extraction and purification protocol depends on the source material, desired DNA yield and purity, and intended downstream application. Several commercially available kits offer user-friendly methods for various sample types. However, the versatile phenol-chloroform extraction technique remains a valuable tool for researchers due to its high DNA yield and cost-effectiveness, although it requires more experience and involves hazardous chemicals.

Reagents required DNA isolation.

Cell lysis buffer:

1. 10 mM Tris-Cl (pH 8.0)
2. 1 mM EDTA (pH 8.0)
3. 0.1% (w/v) SDS

Ethanol, Isopropanol, phenol: chloroform: isoamyl alcohol

Potassium acetate solution

• 60 ml of 5 M potassium acetate
• 11.5 ml of glacial acetic acid
• 28.5 ml of H₂O

Enzymes and Buffers

• DNase-free RNase (4 mg/ml)
• Proteinase K (20 mg/ml).

Chromosomal DNA isolation overview

1. Prepare the bacterial strain for lysis
   1. DH5α (bacterial strain) is cultured in media and incubated overnight.
   2. Transfer 1.5 ml from an overnight culture to a microfuge tube.
   3. Pellet the cells by centrifuging for 1 minute in at room in a microfuge.
   4. Remove the culture medium by aspiration and resuspend the pellet in 50µl of STE buffer
2. Add ~50 µl of acid-washed glass beads to each tube containing the resuspended yeast. Add 20 μl of TE (pH 7.6) to each tube.
3. Add 60 µl of phenol: chloroform: asoamyl alcohol, cap the tubes and mix the organic and aqueous phases by vortexing for 1 minute.
4. Centrifuge the tubes at maximum speed for 5 minutes at room temperature in a microfuge.
5. Transfer the upper aqueous phase to a fresh microfuge tube. Collect the DNA by standard precipitation with ethanol for 15 minutes at 0°C.
6. Recover the precipitate of nucleic acids by centrifugation at maximum speed for 10 minutes at 4°C in a microfuge
7. Remove the supernatant by aspiration and rinse the pellet with 100 µl of 70% ethanol in H₂O. Centrifuge the tubes at maximum speed for 1 minute at room temperature in a microfuge.
8. Remove the supernatant by aspiration and allow the pellet to dry in the air for 15 minutes. Redissolve the pellet in 40 µl of TE (pH 7.6)

Protocol for DNA Extraction and Purification

Purpose

Phenol-chloroform extraction remains a valuable tool for DNA isolation, particularly when high yield and cost-effectiveness are priorities. But for situations requiring faster turnaround or when dealing with hazardous materials is a concern, other methods like spin column purification might be preferred

Materials

• Ammonium acetate, concentrated solution (5M – 7.5M)
• 25:24:1 phenol/chloroform/isoamyl alcohol solution, pH 7.8-8.2
• 24:1 chloroform/isoamyl alcohol solution
• Elution buffer (10 mM Tris-HCl, pH 8.5)
• Glycogen, 20 mg/mL (molecular biology grade)
• 100% Ethanol
• 80% Ethanol

Procedure

1. Add 200 µL of starting material (lysate) into Tube 1. Bring the total volume to 200 µL with the elution buffer if needed.
2. Add 200 µL of the phenol/chloroform/isoamyl alcohol solution into Tube 1.
3. Vortex Tube 1 for 30-60 seconds.
4. Centrifuge Tube 1 at 16,000xg for 5 minutes at room temperature.
5. Remove the top aqueous layer from Tube 1 and transfer it into a new clean tube, Tube 2.
6. Add 200 µL elution buffer into Tube 1.
7. Repeat steps 3 and 4.
8. Remove the top aqueous layer from Tube 1 and transfer it to Tube 2.
9. Add equivalent volumes of the 24:1 chloroform/isoamyl alcohol solution into Tube 2.
10. Vortex Tube 2 for 30-60 seconds.
11. Centrifuge Tube 2 at 16,000xg for 5 minutes at room temperature.
12. Remove the top aqueous layer from Tube 2 and transfer it into a new clean tube, Tube 3.
13. Add ammonium acetate to Tube 3 until the final concentration is 0.75 M.
14. Add 1 µL of the glycogen mixture into Tube 3. Pipette up and down repeatedly to mix.
15. Add 100% ethanol at 2.5 times the volume into Tube 3.
16. Centrifuge Tube 3 at max force for 20 minutes at 4°C.
17. Remove the supernatant without disturbing the pellet.
18. Wash by adding 300 µL of 80% ethanol into Tube 3. Pipette up and down to resuspend the pellet.
19. Centrifuge tube at max force for 15 minutes at 4°C
20. Remove the supernatant without disturbing the pellet.
21. Repeat steps 15 to 17 for a second 80% ethanol wash.
22. Spin Tube 3 on a tabletop centrifuge and remove residual ethanol with a P20 pipette.
23. Let air dry for several minutes
24. Dissolve the pellet in elution buffer or DNase-free molecular-grade water
    • Keep on ice if measuring concentration in the sample. Otherwise, store at -20°C. Avoid multiple freeze-thaw cycles to preserve sample integrity