Preparation of DNA injection mix.

Note that the discussion below is for when a transposase encoding plasmid is being used in combination with the transposon.  Now that we have switched to a delta 2,3 line (Fall 99), there is no need to include the transposase encoding plasmid.  I've provided an abbreviated procedure for preparing an injection mix containing only the transposon (go here).


Before making the injection mix, be certain the DNA is of good quality and free of any insoluble material. The latter will plug the needles. I recommend diluting the DNA samples to a concentration of 500 ng/ul or less and then microfuging the DNA for 5’. I would do this regradless of how the DNA was prepared. Transfer the DNA to a new tube, leaving behind any debris that is pelleted on the bottom of the tube. Check the DNA concentration with a fluorometer.

There are numerous transposase plasmids available, but the one we have been using successfully is one called "p13wc". This was obtained from Susan Abmayr and was constructed in the Maniatis lab. Its exact construction is not known but it shares restriction fragments with other transposase plasmids like pp25.7wc. We have a large preparation of p13wc that should keep us going for many years.

I prepared my DNA for injections by subjecting them to a spermine precipitation followed by two ethanol precipitations. First, I rinsed a 1.7 ml tube with distilled water to remove any particulates that might later clog the injection needle. 300 ul of 100 mM NaCl, 10 mM Tris pH 8, 1 mM Na2EDTA was added to the tube. Then I added 12.5 ug of the transposon (CaSpeR derivative) and 2.5 ug of p13wc. The p13wc is located in a rack in Lakshmi’s freezer labelled "p13wc from Abmayr". Its concentration is 2.5 ug/ul. The solution is gently mixed and then 15 ul of 100 mM spermine is added. Mix again and place the solution on ice for 15’.

The DNA is microfuged for 15’ in the cold. Carefully remove the supernatant by keeping the pipette tip on the side opposite from where the DNA is pelleted. It is unlikely that you will be able to see the DNA precipitate. Dissolve the DNA in 100 ul of 0.5 M NaCl. Dissolve the DNA at room temperature by intermittently tapping the tube throughout a 10’ period. Spermine precipitates are notoriously slow to dissolve and the mechanical agitation helps the process.

After dissolving the DNA, add 100 ul of water and 400 ul of 100% ethanol. Mix thoroughly by inverting the tube several times and place the solution on ice for 15’. Microfuge for 15’ in the cold and discard the supernatant. The DNA should be evident on the side of the tube as distortions in the ethanol layer that sheds from the surface of the tube as you remove the supernatant. Add 50 ul of 0.5 M NaCl and 50 ul of water to the tube. Gently agitate the tube at room temperature for about 5’. Add 200 ul of cold 100% ethanol and mix. If you look closely, you may see a white fiber of DNA precipitate. Ice for 15’ and then microfuge for 15’. Remove the supernatant and rinse the precipitated with 100 ul of 70% cold ethanol. Microfuge the sample for 2’, remove the supernatant, and allow the DNA to air dry.

Once the DNA has dried, dissolve it in 20 ul of injection buffer (do not include the green dye, see note below). Injection buffer consists of 0.1 mM Sodium Phosphate pH 6.8, 5 mM KCl. The injection buffer should be filter sterilized but be certain to rinse water through the filter first in order to rinse wetting agents from the filter. Originally, I had intended to dilute this DNA with injection mix so the final concentration of the transposon plasmid was 250 ng/ul and the transposase plasmid was 50 ng/ul. After quantifying the DNA (in the 20 ul volume) with the fluorometer, I decided not to dilute it further because I had read elsewhere that higher concentrations of DNA could also be injected. Instead, I did one set of injections with a final DNA concentration of 530 ng/ul and the other set of injections with a final DNA concentration of 680 ng/ul. These final concentrations were what the DNAs happened to be at when dissolved in 20 ul. Several other successful transformations have been done with the concentration of DNA at 400 ng/ul. The DNA samples were frozen at -20 when not in use and stored on ice when I took them down to the injection room. I am concerned about nucleases because the injection mix contains no EDTA. Finally, do not add any of the green dye. Hongbing has an indication that the green dye might impair the viability. Moreover, you will be able to see the DNA solution enter the embryos during the injection if you watch closely.