Cesium Chloride: DNA purification for microinjection of mouse embryos
This protocol has been taken from "Manipulating the Mouse Embryo". Hogan et al. Cold Spring Harbor Press. 1994. Although the procedure is laborious, it results in DNA samples that are easily microinjected and should yield uniformly high frequencies of embryo survival and transgenesis. To avoid accumulation of dust during the concentration of DNA, all solutions should be filtered through a 0.2 m filter and all tubes and pipettes should be rinsed with filtered water prior to use. Do not use pipette tubes packed by hand in the lab, obtain commercial tips for this procedure.
1. Isolate 50g or more of the DNA fragment to be microinjected. Excise the band from the agarose gel in a minimum volume of agarose and electroelute the DNA from the gel slice in a dialysis bag for 2-3 hours. Carefully remove the buffer and agarose from the bag (most of the DNA will adhere to the bag), add fresh buffer and reverse the polarity of electroelution for a few minutes to recover the DNA.
2. Extract DNA several times with phenol and then extract with ether three times to remove residual phenol.
3. Precipitate with ethanol several times and resuspend in a minimum volume (e.g. 50-100l) of injection buffer (T low E - 10mM TRIS, pH 7.5, 0.1mM EDTA - available from the Transgenic Core Lab). Determine DNA concentration.
4. Dissolve at least 10g of DNA in 2.4 ml of 10mM TRIS-HCl (pH 8.0), 1mM EDTA. Add exactly 3.0g ultrapure CsCl and dissolve. Check that density is 1.700.01g/ml. Adjust if necessary.
5. Transfer the DNA solution to a 1.3cm polyallomer ultracentrifuge tube, cover with light paraffin oil, and centrifuge in a SW50.1 rotor at 200C for 48 hours at 40,000 rpm.
6. Collect 0.2ml fractions from the bottom of the tube. Assay ~8 fractions from the middle of the tube by running 1-3l on an agarose minigel and pool the fractions containing DNA.
7. Dialyze at 40C against a large volume of injection buffer, changing the injection buffer several times over a 48 hour period.
8. Filter through a 0.2m filter; determine DNA concentration; dilute to 100ng/l in embryo tested injection buffer. Run a gel to confirm purity and concentration.
