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ZIRC
This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.

CHAPTER 9 - MOLECULAR METHODS

Total Nucleic Acid Extraction Procedure for Zebrafish Embryos

(Source: D. Ellies)

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

Buffer: use RNase free reagents!
  • 100mM Tris-HCl (pH 8.0)
  • 100 mM EDTA
  • 250 mM NaCl
  • 1% SDS
  • You do not need to remove embryos from their chorions.

    For a single embryo:

    1. Rinse embryo in sterile water.
    2. Homogenize in 10µl of buffer by hand with a sterile pipette tip.
    3. Add 90µl of buffer and homogenize again.
    4. Extract with 50µl of phenol:chloroform:isoamyl-alcohol (50:48:2). Mix gently by inversion, centrifuge and collect top aqueous phase. 5. Extract aqueous phase as above but with 50µl of chloroform:isoamyl-alcohol (24:1).
    6. Add 25µl of NaAcetate 3M pH 7 and precipitate with 200µl of ethanol.
    7. Resuspend pellet in 20µl of TE.

    For 5 or more embryos:

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