Methods/DNA barcoding

DNA extraction using HotShot

Mixing the buffers

Alkaline Buffer (= 25 mM NaOH + 0.2 mM disodium EDTA)

1. Premix 10M NaOH:
   • 70 ml H₂O + 40g NaOH and adjust to 100 ml

   or

   • 17.5 ml H₂O + 10g NaOH and adjust to 25 ml

1. To 25 ml of H₂O add:
   • 62.5 μl of 10M NaOH
   • 10 μl of 0.5 M disodium EDTA

Neutralizing Buffer (40 mM trisHCl)

Mix the following:

• 24 ml of H₂O
• 1 ml of 1M trisHCl

---

1. Premix 10M NaOH:
   • 70 ml H₂O + 40g NaOH and adjust to 100 ml

   or

   • 17.5 ml H₂O + 10g NaOH and adjust to 25 ml
2. To 25 ml of H₂O add:
   • 62.5 μl of 10M NaOH
   • 10 μl of 0.5 M disodium EDTA

Extraction

• 10–15 μl of alkaline buffer + specimen
• Incubate at 65°C for 18 mins + 98°C for 2 mins
• Add the same amount of neutralizing buffer