Extraction of Genomic DNA CTAB-Lysozyme Method

Isolation of genomic DNA is necessary for PCR and Southern analysis. The CTABLysozyme method of genomic DNA extraction yields good quality DNA. Typically, the method yields good quality DNA that is suitable for Southern and PCR but not applications that require larger DNA fragments (>50 kb) such as pulse-field gel electrophoresis. The CTAB method is also suitable for smaller culture volumes (10 mL) and is useful when screening a number of clones. See also Connell et al (Connell 1994). A. Materials and Reagents 1. Glycine, 10% (w/v) 2. GTE solution 3. Lysozyme solution 4. SDS, 10% 5. Proteinase K, 10 mg/mL 6. NaCl, 5M 7. CTAB solution 8. Chloroform:isoamyl alcohol (24:1) 9. Isopropanol 10. Ethanol, 70% 11. TE Solution 12. 15 mL conical tubes 13. Microfuge tubes 14. Tabletop centrifuge 15. Microcentrifuge B. Protocol Steps 1. Optional: 24 hours before harvesting cells for genomic DNA preparation, add glycine to a late log culture to a final concentration of 1% using a 10% (w/v) glycine stock. Incubate at 37C (the glycine weakens the cell wall and for some strains will lead to a higher yield of DNA). 2. Transfer 10 ml of culture to 15 ml conical tube and spin in tabletop centrifuge at 2,000 x g for 20 minutes. 3. Discard supernatant and resuspend cell pellet in 1 ml GTE Solution. Transfer to 2 ml microfuge tube. Centrifuge for 10 minutes. 4. Discard supernatant and resuspend cell pellet in 450 l GTE Solution. Add 50 l of a 10 mg/ml lysozyme solution. Mix gently and incubate at 37C overnight. 5. Add 100 l 10% sodium dodecyl sulfate and mix gently. Add 50 l 10 mg/ml Proteinase K (Sigma, cat. P4914) and mix gently. Incubate at 55C for 20 to 40 minutes. 6. Add 200 l 5M NaCl and mix gently (NaCl blocks the binding of DNA to Cetrimide). 7. Preheat CTAB solution to 65C. Add 160 l and mix gently. Incubate at 65C for 10 minutes.

8. Add an equal volume (~1 ml) chloroform:isoamyl alcohol (24:1), shake gently to mix, and spin in microfuge for 5 minutes. 9. Transfer 900 l of aqueous layer to a fresh microfuge tube. 10. Repeat extraction with chloroform:isoamyl alcohol (24:1), shake gently to mix, and spin in microfuge for 5 minutes. 11. Transfer 800 l to fresh microfuge tube. Note: For BSL3 organisms: dip tube in a disinfectant such as VespheneII to disinfect outer surface --supernatant can be processed in BSL2 lab. 12. To 800 l aqueous layer, add 560 l (0.7X vol) isopropanol, mix gently by inversion until the DNA has precipitated out of solution. Incubate at room temperature for 5 minutes. Spin in microfuge for 10 minutes. 13. Aspirate supernatant and add 1 ml 70% ethanol to wash DNA pellet. Mix gently by inversion and spin in microfuge for 5 minutes. 14. Aspirate supernatant and air-dry DNA pellet for 15 minutes. Do not overdry. Add 50 L TE buffer to dried DNA pellet and store at 4C overnight to allow pellet to dissolve.