DNA extraction - Salting Out: Difference between revisions

#Add 5μL Proteinase K to each mL of Digestion Buffer

#Homogenise (or simply place) tissue in solution

#Incubate at 55°C for 1 hour to overnight

#Centrifuge at maximum speed in a benchtop centrifuge for 2 minutes

#Transfer supernatant into a new tube  

#Add 1/10 volume of Sodium Acetate 3M (pH5.2)

#Invert to mix and incubate at -20°C for ~30 minutes

#Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes

#Transfer supernatant to a new tube

#Add >2 volumes of 98% ethanol

#Invert to mix and incubate at -20°C for 30 minutes

#Centrifuge (preferably at 4°C) at maximum speed in a benchtop centrifuge for 20 minutes

#Wash pellet with 70% ethanol, dry and resuspend in water or TE

#After adding sodium acetate and centrifuging be careful not to transfer any of the white solid (SDS) into the fresh tube

#Ensure to dry the pelletted DNA completely before attempting to resuspend

#This protocol has evolved with me and I am sure many others, please share your optimisations.

#You may wish to kill the Proteinase K (95°C, 10 minutes) after the extraction though if you are proceding straight to PCR (and don't leave your reactions sitting around before thermal cycling) the denaturation step will take care of that

#Damien Broderick from Queensland Depaqrtment of Primary Industries and Fisheries supplied me with his version of this protocol to start me off.