DNA Extraction Protocol

This protocol was optimized for extraction of long reads from oyster microbiomes. Modified from Suter et al. 2017.

Reagents

• Lysozyme
• Proteinase K solution
• Sodium dodecyl sulfate (20%)
• Phenol:Chloroform:Isoamyl alcohol (25:24:1; pH 8.0)
  • Prepare by adding supplied buffer, shake well, allow phases to separate and check pH of buffer using pH paper. Add hydroxyquinoline to a final concentration of 0.1% (100 mg hydroxyquinoline per 100 ml phenol:Chloroform:IAA).
• Chloroform:Isoamyl alcohol (24:1)
• TE buffer
• 0.5 M EDTA
• 1 M Tris (pH 8.3)
• Sucrose
• Sterile water
• Amicon Ultra-4 w/ 100 kDa MWCO Centrifugal Filter Unit

Prepare lysis buffer (adjust volumes according to sample number):

Filter sterilize or autoclave before use

Final Concentration	For 20 ml:
40 mM EDTA	1.6 ml of 0.5 M EDTA
50 mM Tris (pH 8.3)	1.0 ml of 1 M Tris (pH 8.3)
0.73 M Sucrose	5.13 g Sucrose
	Sterile water to 20 ml

Prepare enzymes (adjust volumes according to sample number):

• Lysozyme solution: 2 mg lysozyme per 40 µl Lysis Buffer
• Proteinase K solution: 1 mg Proteinase K per 100 µl Lysis Buffer

Extraction:

1. Thaw frozen samples and shuck. Add tissue to a 50ml conical tube.

2. Add 40 µl lysozyme solution to the sample.

3. Incubate at 37°C rotating for 45 minutes.

4. Add 100 µl proteinase K solution and 100 µl 20% SDS. Recap.

5. Incubate at 55°C while rotating for 2 hours.

6. Add 3ml Phenol:Chloroform:Isoamyl alcohol. Vortex 10 seconds.

7. Spin for 5 minutes at 3500xG.

8. Carefully transfer aqueous phase (crude DNA extract) to new 15 ml falcon tube and add 3 ml Chloroform:Isoamyl alcohol. Vortex 10 seconds. Spin for 5 minutes at 2500xG.

9. Prepare column by washing with 1ml TE. Centrifuge for ~5 min @ 2000g.

10. Carefully transfer aqueous phase from [10] to an Amicon filter. Concentrate to ~100 µl final volume (~10 min @ 2000XG for above filter. Spin longer if necessary.).

11. Wash by adding ~4 ml TE buffer to filter and re-spin to final desired volume.

12. Recover any remaining DNA off filter by rinsing with another 50 μl TE and pooling with volume from above.