PROTOCOL 004: Zebrafish Genomic DNA Purification





100 mM NaCl

50 mM Tris pH 8

50 mM EDTA pH 8                                                                              

1% SDS


Proteinase K                                                                                       

20 mg/mL in water


Phenol-Chloroform-Isoamyl (PCI)                                                       

25:24:1 (Amresco, K169)



70% ethanol

Long Pasteur pipettes

1X TE pH 8




Euthanize zebrafish by Tricaine overdose


Add 100 mL NTES and 5 mL Prot-K and rock for 2 – 5 hours or overnight at 55 C


Add 100 mL PCI and rock gently at room temperature (RT) for 30 – 60 minutes


Centrifuge 5 minutes at 14k rpm at RT


Carefully remove aqueous (upper) phase and place in clean container (avoid disturbing interphase); discard PCI in approved container.


Add 3 vol of RT isopropanol, cap and invert a few times – you should see DNA ppt as a 'fuzzy' ball.


Spool DNA using a fire-sealed Pasteur pipette


Dip DNA-laced pipette in isopropanol followed by 70% ethanol wash


Let DNA to dry for about 2 – 3 minutes and break off end of pipette with diamond tip into microfuge tube


Add 50 mL 1X TE and incubate at 37 C for 30 minutes


Determine concentration and adjust to 0.1 mg/mL with TE