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
2. 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

How to use the buffers to extract DNA

1. Place the specimen into 10–15 μl of alkaline buffer in a microvial
2. Incubate the sample at 65°C for 18 mins + 98°C for 2 mins in a PCR machine
3. Add the same amount of neutralizing buffer as you used of the alkaline buffer (so 10 μl alkaline + 10 μl neutralizing or 15 μl alkaline + 15 μl neutralizing)

Our extraction protocol

BEFORE starting, check if we have:

• enough buffers (alkaline and neutralizing) – if not, mix new one
• enough sterile stripes – if not, autoclave more

Prepare the Extraction Plate Map (MS Excel)

• select the samples you will extract
• make a plate map, keep at least 2 positions empty
• save the plate map, and print it out
• mark positions with BIG animals (they have "yes" in the sample sorting file)
• upload the plate map into the Lab Cloud

Prepare the place for work

• 2× 48-well plastic tray, fill each well with a bit of purified water
• burner with enough alcohol
• lighter
• sterile Petri dish with a bit of purified water
• paper tissues
• one soft and two hard forceps (sterilize properly in the beginning)

Prepare stripes

1. Prepare 12 sterile strips.
2. Pipette 15 μl of alkaline buffer into each microvial (use tips with filter)
3. Close all strips.
4. Number the strips – numbers 1–12 on the lid of the first microvial in the strip
5. Mark the empty positions.

Put specimens into the alkaline buffer

• Take one specimen from each sample, put in 48-well plate in purified water
• In large specimens, take 1–3 legs (ideally each from one side) using hard forceps. Return the specimen to the original vial, and mark the vial label in red
• After some time (5–15 mins) move the specimen from the water to the strip microvial with buffer using sterile forceps.

DNA extraction

1. Put into PCR machine – the program is called HOTSHOT (65°C for 18 mins + 98°C for 2 mins)
2. Add 15 μl of neutralizing buffer into each microvial.

After DNA extraction

• Pipette out the liquid into new marked strips. This will be your DNA extract
• Add 75% or 95% alcohol to the strips with specimens
• Add Sample ID labels to each sample.

Storing the extraction

• Double-check that the plate is properly marked (e.g., Extraction: Eric001)
• You can use it immediately for PCR
• If you will use it soon (in 1–2 days), keep it in the -20°C freezer
• If you will not use it soon or it is finalized, keep it in the -70°C freezer