HotSHOT genomic DNA extraction

Prepare the BASE solution (50X):

• 14.03g KOH crystals (1.25M final concentration)
• 4mL of 0.5M EDTA (10 mM final concentration)
• ddH2O to 200mL total volume

Prepare the NEUTRALISATION solution (50X)

• 63.04g Tris-HCL (2M final concentration); also called Trizma HCl
• ddH2O to 200mL total volume

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

If larvae: add larvae one per well with Pasteur pipette. Take up all the liquid using a P200 pipette.

If finclip: add finclip directly to well (this can be done while finclipping).

Add 50 µL 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 50 µL 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 sealed plate

or

-20°C for long-term storage. This will also prevent the samples from evaporating.