May 19, 2023

Public workspaceHotSHOT genomic DNA extraction V.2

DOI
dx.doi.org/10.17504/protocols.io.j8nlkw945l5r/v2
  • FishFloorUCL1,
  • FishFloorUCL1
  • 1University College London
Francois Kroll
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.j8nlkw945l5r/v2
Protocol Citation: FishFloorUCL, FishFloorUCL 2023. HotSHOT genomic DNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw945l5r/v2Version created by Talia Pittman
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: March 31, 2023
Last Modified: May 19, 2023
Protocol Integer ID: 79829
Keywords: DNA, extraction, HotShot, danio rerio, zebrafish
Abstract
How to extract genomic DNA from larvae or finclips using the HotSHOT method.
Materials
Materials
Prepare the BASE stock solution (50X):
  • Amount14.03 g KOH crystals (1.25M final concentration)
  • Amount4 mL of 0.5M EDTA (10 mM final concentration)
  • ddH2O to Amount200 mL total volume
Can be stored at room temperature for up to four years
Prepare the NEUTRALISATION stock solution (50X)
  • Amount63.04 g Tris-HCL (2M final concentration), also called Trizma HCl
  • ddH2O to Amount200 mL total volume
Can be stored at room temperature for up to four years
Procedure
Procedure
30m
30m
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.

Note
(Finclips) Transfer directly to a 96-well plate during fin-clipping
(Larvae) After culling:
  • Pipette technique: Use a pipette set to 5µL to transfer larvae with as little fish water as possible
  • Forceps technique: Transfer individual larvae using blunt round-ended forceps. This technique is only possible if you have already loaded the plate with 50µL 1X BASE solution (step 5)


Add Amount50 µL of 1X BASE solution into each well of the plate

Seal the plate and place on PCR block Temperature95 °C Duration00:30:00
30m
Cool at TemperatureRoom temperature

Add Amount50 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.
Storage
Storage
Store at Temperature4 °C if you will use the DNA in the next few weeks. Beware, the samples will slowly evaporate from a sealed plate. Alternatively, Temperature-20 °C for long-term storage. This will also prevent the samples from evaporating.