Team:Glendale CC AZ/Protocols/DNAIsolation

Glendale Community College Arizona

Protocols

Genomic DNA Isolation Protocol

Purpose: Isolate and purify genomic DNA from a Deinococceae species bacterium.

Materials

  -1.5ml microcentrifuge tubes 
  -water bath, 80°C 
  -water bath, 37°C 
  -isopropanol, room temperature 
  -70% ethanol, room temperature 
  -water bath, 65°C  
  -50mM EDTA 
  -10mg/ml lysozyme  
  -10mg/ml lysostaphin 

Procedure

• • Note: Initial sonification step was used for D. rad. instead of lysozyme**

1. Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube.

2.Centrifuge at 13,000–16,000 × g for 2 minutes to pellet the cells. Remove the supernatant. For Gram Positive Bacteria, proceed to Step 3. For Gram Negative Bacteria go directly to Step 6.

3.Resuspend the cells thoroughly in 480μL of 50mM EDTA.

4.Add the appropriate lytic enzyme(s) to the resuspended cell pellet in a total volume of 120μl, and gently pipet to mix.

5.Incubate the sample at 37°C for 30–60 minutes. Centrifuge for 2 minutes at 13,000–16,000 × g and remove the supernatant.

6.Add 600μL of Nuclei Lysis Solution. Gently pipet until the cells are resuspended.

7.Incubate at 80°C for 5 minutes to lyse the cells; then cool to room temperature.

8.Add 3μL of RNase Solution to the cell lysate. Invert the tube 2–5 times to mix.

9.Incubate at 37°C for 15–60 minutes. Cool the sample to room temperature.

10.Add 200μL of Protein Precipitation Solution to the RNase-treated cell lysate. Vortex vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution with the cell lysate.

11.Incubate the sample on ice for 5 minutes. 12. Centrifuge at 13,000–16,000 × g for 3 minutes.

12.Centrifuge at 13,000–16,000 × g for 3 minutes.

13.Transfer the supernatant containing the DNA to a clean 1.5ml microcentrifuge tube containing 600μL of room temperature isopropanol.

Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid in the tube to avoid contaminating the DNA solution with the precipitated protein.

14.Gently mix by inversion until the thread-like strands of DNA form a visible mass.

15.Centrifuge at 13,000–16,000 × g for 2 minutes.

16.Carefully pour off the supernatant and drain the tube on clean absorbent paper. Add 600μL of room temperature 70% ethanol and gently invert the tube several times to wash the DNA pellet.

17.Centrifuge at 13,000–16,000 × g for 2 minutes. Carefully aspirate the ethanol.

18.Drain the tube on clean absorbent paper and allow the pellet to air-dry for 10–15 minutes.

19.Add 100 μL of DNA Rehydration Solution to the tube and rehydrate the DNA by incubating at 65°C for 1 hour. Periodically mix the solution by gently tapping the tube. Alternatively, rehydrate the DNA by incubating the solution overnight at room temperature or at 4°C.

20.Store the DNA at 2–8°C.

• Special Thanks to Beau Grothendick for this protocol*