PROTOCOL 008: GENOMIC DNA ISOLATION FROM ZEBRAFISH EMBRYOS

(10/2012)

 

REAGENTS

 

NTES (100 mL)                       

10 mM Tris, pH 8.0                 

25 mM EDTA

50 mM NaCl

0.5% v/v SDS

 

Proteinase K (protK)                      

10 mg/ml

 

Phenol/Chloroform/Isoamyl (PCI)

 

1-Bromo-3-chloropropane (BCP)             

 

EtOH               

75%

 

Isopropanol

                                                                       

Clipped yellow tips                                    

 

1X TE

 

 

PROCEDURE

 

Collect approximately 50 embryos that have developed ³ 24h into a 1.5 ml Eppendorf tube. ItÕs not necessary to dechorionate embryos.

 

Remove embryo media and add 600 ml NTES + 30 ml protK.

 

Place on rocker in 55¡ C incubator and rock overnight.

 

Add 600 ml PCI and rock for 30 minutes at room temperature.

 

Centrifuge tube for 10 minutes at 14k rpm.

 

Carefully remove aqueous phase (upper) using a 200 ml clipped yellow tip and transfer to a clean tube. Discard phenol phase (lower) in appropriate waste container.

 

Add 600 ml BCP to aqueous phase and rock for 10 minutes at room temperature.

 

Repeat centrifugation.

 

Carefully remove approximately 400 ml of the aqueous phase (upper) using a 200 ml clipped yellow tip and transfer to a clean tube. Discard BCP phase (lower) in appropriate waste container.

 

Precipitate DNA by adding 600 ml of isopropanol and inverting tube to mix well. You should see a clump form – it will look like a small ball of yarn – this is DNA.

 

Let DNA settle to the bottom and remove as much of the isopropanol as possible without disturbing DNA.

 

Wash the DNA pellet with 75% EtOH. Remove ethanol as above.

 

Let pellet dry at room temperature for 5 – 10 minutes.

 

Add 200 ml 1X TE.