T. H. (Henry) Hung [email protected], University of Oxford

📬 For any intended reader only

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Reagents (for each sample)

<aside> 💡 Scale up the amount of reagents when working with more samples.

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• [ ] 600 μL CTAB Lysis Buffer (Promega MC1411) / Carlson Lysis Buffer (Fisher Scientific 50-196-447)
  • CTAB Lysis Buffer Recipe
• [ ] 5 μL RNase A (Thermofisher 12091021)
• [ ] 1.2 mL Chloroform-isoamyl alcohol (24:1) (Sigma C0549-1PT) / Chloroform / Phenol-chloroform-isoamyl alcohol (25:24:1)
• [ ] ~600 μL Isopropanol
• [ ] 1 mL 70% EtOH
• [ ] 1.2 μL β-mercaptoethanol (added fresh to the buffer before extraction)

<aside> ⚠️ Once added β-mercaptoethanol into the CTAB Lysis Buffer, start the extraction within an hour. β-mercaptoethanol is a strong reducing agent which prevents oxidation of polyphenols (oxidised polyphenols bind to DNA and can hardly be removed), denatures proteins, and inactivates DNases and RNases.

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• [ ] 30–100 μL TE Buffer (Thermofisher 12090015)

Part 1: DNA extraction

⏱️ 1.5-hour hands-on time ⏱️ 2-hour procedure

<aside> 💡 Fresh, actively growing foliage has the most DNA and the least contaminants. DNA quality is the best when the foliage is immediately frozen or quickly dried in silica gel after sampling. Store the samples in –20°C or –80°C. For starting material, use 100–150 mg of fresh or frozen tissue, or approximately 20–30 mg or 2 cm$^2$ of dried material.

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<aside> ☠️ CTAB is very toxic to the environment. Chloroform is extremely volatile and toxic. Perform the experiment in a fume hood and discard the waste appropriately.

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<aside> 🌡️ Set the water bath / heat block to 65°C.

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