HotSHOT genomic DNA extraction

Prepare the BASE stock solution (50X):

• 14.03g KOH crystals (1.25M final concentration)
• 4mL of 0.5M EDTA (10 mM final concentration)
• ddH2O to 200mL total volume Can be stored at room temperature for up to four years

Prepare the NEUTRALISATION stock solution (50X)

• 63.04g Tris-HCL (2M final concentration), also called Trizma HCl
• ddH2O to 200mL total volume Can be stored at room temperature for up to four years

Prepare fresh 1X BASE and 1X NEUTRALISATION solution in nuclease-free H2O.

Transfer finclips or culled larvae to individual wells of a 96-well plate.

Add 50µL of 1X BASE solution into each well of the plate

Seal the plate and place on PCR block 95°C 0h 30m 0s

Cool at Room temperature

Add 50µL of 1x NEUTRALISATION solution into each well of the plate.

Note, the extracted DNA concentration is often too high for downstream applications like PCR or KASP.

Store at 4°C if you will use the DNA in the next few weeks. Beware, the samples will slowly evaporate from a sealed plate. Alternatively, -20°C for long-term storage. This will also prevent the samples from evaporating.