The purification of DNA fragments for microinjection is extremely important. Linear DNA fragments integrate much more efficiently than circular forms, and prokaryotic vector sequences can reduce the efficiency of transgene expression. Contamination of the DNA with impurities (e.g., phenol, ethanol, or detergents) can adversely affect the survival of injected eggs, and particu-late matter can block needles during microinjection (see Note 12).
A simple purification method is described here for isolating DNA fragments from agarose gels using P-Agarase I (New England Biolabs), although GELase (Epicenter Technologies, Cambridge, Cambs, UK) is equally suitable.
1. Excise the reporter construct to be microinjected from the vector by digestion with the appropriate restriction enzyme(s) according to the conditions recommended by the supplier.
2. Fractionate 5-20 |g of the digested DNA by agarose-gel electrophoresis using 1X TBE buffer containing 0.5 |g/mL ethidium bromide (see Note 13).
3. Wearing appropriate protective wear, view the gel on a long-wave transilluminator, and using a scalpel blade, cut a large well in front of the fragment band to be purified. Place the gel at 4°C, and fill the well with molten (45-50°C) LMP agarose. Allow to solidify, and then return the gel to the electrophoresis tank until the band of interest has migrated into the LMP agarose. Using a clean scalpel blade excise the band, and place in a clean microcentrifuge tube (see Note 14).
4. Incubate the sample at 65°C until molten (approx 10 min) After estimating the sample volume, add 1/10 vol of 10X Agarase buffer, mix, and equilibrate at 40°C.
5. Add 2 U of P-Agarase I/200 |L of 1% (w/v) LMP agarose. Mix well and incubate for 1 h at 40°C (see Notes 15 and 16).
6. Extract the sample twice with phenol, twice with chloroform, and once with diethyl ether, each time retaining the aqueous phase (see Notes 17 and 18).
7. Transfer the aqueous phase to a fresh tube, and precipitate with 0.6-1X vol of propan-2-ol. Pellet the DNA by centrifugation at 15,000g for 20 min. Wash the pellet thoroughly with 70% (v/v) ethanol, and allow to dry at room temperature.
8. Dissolve the DNA pellet in microinjection buffer, and pass through a 0.22-pm filter (Spinex, Costar, High Wycombe, Bucks, UK). Store frozen at -20°C.
9. The concentration and integrity of your purified DNA fragment can be accurately judged by agarose gel-ethidium bromide electrophoresis against samples of known amount (see Note 19).
10. Prior to microinjection, DNA is diluted to a concentration of 1-5 pg/mL with filtered microinjection buffer (see Note 20).
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