Protocols

Purification of DNA for pronuclear microinjection

At least 2 mg in 50 ml of a DNA construct should be supplied as a single linear DNA fragment prepared as described below. While a variety of other methods are available to prepare DNA, we recommend that you use this procedure.

IMPORTANT NOTE: The LMP agarose should be from Gibco/BRL (cat#15517-014) as some other brands of agarose are toxic and kill the embryos.

The source of the GELase is Epicenter Biotechnology (1-800-284-8474; GELase cat# G09050; Buffer cat # G191ML). You may follow the Epicenter GELase protocol exactly, or use the following protocol or you can use Promega's AgarAce.

1. Digest at least 20 mg of DNA with appropriate enzymes to completely remove vector sequences (total volume of, say, 100 ml, with 5ml or so of each enzyme needed). Digest 1 hour to overnight. Add 40 ml 6X loading buffer (we use a Ficoll based buffer), mix completely. The exact amount to digest depends upon the resolution of vector from insert in the gel, gel well volumes, etc. -- use your experience and common sense!
2. Run digested DNA on appropriate percentage (usually 0.7-1.0% for transgenic constructs) LMP agarose gel (Gibco/BRL cat. # 15517-014). Load into several individual wells (10 x 20 ml samples), or in one continuous well (about 1 mg DNA per cm width of comb). Run gel slowly in 1X TAE; run at 60 volts) for several hours.
3. On UV light box, quickly cut out appropriate bands trying to trim away as much agarose as possible. Keep bands away from UV light for as long as possible -- minimized exposure to UV increases cloning efficiency and reduces DNA damage. Put gel slices into eppendorf tube. Volume should be about 500 ml. If necessary, split bands equally into 2 tubes.
4. Place capped tube in 65°C water bath for 10 min., vortex well (but quickly), reincubate at 65°C for 10 min., vortex and place tube at 40-42°C for 10 min.
5. Add "50X" GELase buffer to melted agarose pieces to make it 1X final concentration.
6. Add 2 ml GELase/500 ml volume. Incubate overnight at 40-42°C.
7. To check GELase digestion, place tube on ice for 10 min. -- if still liquefied, proceed. If there are any indications of gelling or viscosity, remelt as above (step 4) and repeat GELase treatment for several hours.
8. Perform two phenol extractions with equal volume (about 400 ml) at room temperature, properly equilibrated fresh phenol (first interface will have material deposited: second should not. If it does, do one extra phenol extraction). Do one phenol:chloroform:isoamyl alcohol extraction (25:24:1) and one fresh chloroform extraction. NOTE: Even though there are a lot of extractions here, by careful pipetting and recentrifuging interface components into the conical tip of the eppendorf tube, you should still be left with 95% of your solution (and therefore DNA) at the end of this procedure.
9. Add slightly more than 1/10 volume of 3.0 M sodium acetate (pH~5.2), e.g. if you have 400 ml of solution, add 50 ml. Then add two volumes of 100% EtOH (fill tube, essentially), mix well, let sit at room temperature 10 min., and microcentrifuge 15-30 min. to recover DNA.
10. Rinse pellet with 500 ml 70% EtOH (room temp.), dry pellets, and resuspend in TE.
11. Reprecipitate DNA by adding "1/10 volume" 3.0 M sodium acetate (pH 5.2), and at least 2 volumes of 100% EtOH. Precipitate as you are used to doing (maybe use dry ice for 15 min., or O/N at -20°C, and recover by centrifugation - full speed microfuge for 15-30 min). Rinse pellet with 70% EtOH (room temp.), respin 5 min., aspirate S/N, wash again with 70% EtOH. Respin, aspirate, dry. Redissolve in injection buffer (10mM Tris pH 7.5, 0.1 mM EDTA; NOTE LOW EDTA CONCENTRATION).
NOTE: Increases in purity/quality of DNA are thought to be achieved if more than one round of 70% EtOH rinsing is performed, or if two or more EtOH reprecipitations are carried out (no phenol extractions: just redissolve the DNA in 400 ml TE, add 1/10 volume sodium acetate, and at least 2 volumes EtOH, then recover as above).
12. Determine OD260. Run an aliquot on a gel adjacent to size markers to determine DNA quality, photograph.
13. Freeze at -20°C and bring the DNA fragment to the Transgenic Mouse / Embryonic Stem Cell Shared Resource.