Chelex DNA extraction Protocol

Make up enough 10% Chelex for entire plate (195µL /individual + slop) (ie. 1g Chelex: 9mL dH2O)

Place a magnetic stir bar in the jar containing your 10% Chelex solution. The Chelex powder is very heavy and needs to be constantly stirred in order to maintain consistency.

Pipette 195µL of Chelex into each well of a 96-well plate.

Add approximately 1mm^2 piece of tissue (usually operculum punch or adipose fin clip) in each well. Be careful when doing this, as it is easy to mess up.

Add 5µL of proteinase K to each well using a repeater pipette. This should always be the last step, as proteinase K will digest itself at room temperature with no substrate.

Cover plate and incubate in the thermocycler under the following conditions: 60°C for 120 min (tissue digestion) 95°C for 15 min (heat kill for proteinase K) This protocol should be saved in most of the thermocyclers (just scroll through). Sometimes this is not sufficient for complete digestion, so a second addition of 5uL of proteinase K followed by a repeat of the incubation procedure may be necessary. Alternatively, you could increase the digestion period from 120 min to 240 min. This is what I usually do and seems to work quite well.

Once digestion is complete, vortex briefly to break up any remaining pieces of tissue, then vortex at 1500 rpm for 2 min to collect Chelex and any other remaining solids on the bottom of the well.

Pipette off the supernatant and add to a new 96 well. This will be your undiluted DNA stock that will be stored for future use. Store at -20°C in chimney well plates with caps.

Make up a 1:10 dilution plate to be used for the assays. Usually 200µL is sufficient to run all assays. Add 180µL of dH2O to each well, and then add 20µL of your undiluted stock. Vortex and spin to finish the procedure. The 1:10 dilution plate can be stored in the fridge to avoid multiple freeze/thaw cycles.