PROTOCOL 004: Zebrafish Genomic DNA Purification

 

REAGENTS

 

NTES                                                                                                  

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)

 

Isopropanol

70% ethanol

Long Pasteur pipettes

1X TE pH 8

 

PROTOCOL

 

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