DNA Isolation Procedure (zebrafish embryos)

1. Collect embryos in a 96-well thermal cycler plate. If the embryos are stored in methanol, distribute 1 embryo per well and then incubate plate at 50°C to eliminate any residual methanol. It is essential to eliminate all methanol before proceding but do not overdry the embryos so that they fall out of the plate.

2. Add 50 µl of lysis buffer. Incubate overnight at 50°C. Make sure the plates are sealed well.

3. If the DNAs will not be used for AFLP or other protocols that require intact double-stranded DNA, incubate samples at 98°C for 10 minutes. Alternatively, the DNAs can be diluted and then incubated at 98°C as described to inactivate the proteinase K.

The final concentration of each sample will be approximately 50 ng/µl.

Lysis buffer

10 mM Tris-HCl, pH 8.3
50 mM KCl
0.3% Tween-20
0.3% Nonidet P40
0.5 µg/µl proteinase K (add fresh before use)

A large bottle of lysis buffer without proteinase K is made up for use as a common stock (storage 4 deg C). To use, add proteinase K to the amount of lysis buffer required to process your samples without contaminating the common stock of buffer. Pour the amount of buffer you need into a 50 ml tube and add 350 µl of 14 mg/ml proteinase K per 10 ml of buffer.