NUCLEIC ACID EXTRACTION PROCEDURE FOR ZEBRAFISH EMBRYOS

By Debbie Ellies, Loeb Institute for Medical Research, Ottawa Civic Hospital, 725 Parkdale Avenue, Ottawa, Ontario, CANADA K1Y 4E9

This rapid, simple procedure yields DNA and RNA which can both be seen on a normal agarose gel. We remove the unwanted nucleic acid with the appropriate nuclease.

Buffer:

• 100mM Tris-HCl (pH 8.0)
• 100 mM EDTA
• 250 mM NaCl
• 1% SDS
• use RNase free reagents!

You do not need to remove embryos from their chorions.

For a single embryo:

• Rinse embryo in sterile water.
• Homogenize in 10ul of buffer by hand with a sterile pipette tip.
• Add 90ul of buffer and homogenize again.
• Extract with 50ul of phenol:chloroform:isoamyl-alcohol (50:48:2). Mix gently by inversion, centrifuge and collect top aqueous phase.
• Extract aqueous phase as above but with 50ul of chloroform:isoamyl-alcohol (24:1).
• Add 25ul of NaAcetate 3M pH 7 and precipitate with 200ul of ethanol.
• Resuspend pellet in 20ul of TE.

For 5 or more embryos:

• Rinse embryos in sterile water.
• Homogenize in 100ul of buffer by hand with a sterile plastic pestle
• Add 300ul of buffer and homogenize again.<
• Extract with 200ul of phenol:chloroform:isoamyl-alcohol (50:48:2).
• Mix gently by inversion, centrifuge, and collect top aqueous phase.
• Extract aqueous phase as above but with 200ul of chloroform:isoamyl-alcohol (24:1).
• Add 100ul of NaAcetate 3M pH 7 and precipitate with 800ul of ethanol. Note: with 5 or more embryos, the nucleic acid precipitate is immediately visible and can be centrifuged right away.
• Centrifuge and resuspend pellet in 20ul of TE.

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