Pronuclear injection to produce transgenic mice

This protocol was developed by Jan Parker-Thornburg at The Ohio State Universtiy


1)Run the DNA digest on an agarose gel:

• Make a 1% Seaplaque (or other very high quality) Low melt agarose gel using 1X TAE prepared according to Maniatis (no EtBr).
• Load the gel with markers on the end separated from the digest of interest by 1 lane. (load the DNA at a concentration of no more than 200 ng/lane--otherwise you run the risk of trapping fragments that you don't want in the band of interest. )
• Run the gel slowly: 30-40 mAmps.
• Once the run is completed, stain the gel with a fairly low concentration of EtBr (about 1�/ml) for 10 min keeping the gel in the dark.
• When finished staining, visualize gel using longwave UV, and cut the band of interest.

2) Purifiying the DNA fragment.

• Load about 200-300� of the gel cut onto a GeneClean spin column(BIO 101'S GeneClean Spinkit)--usually a fragment will require 6-8 columns to take care of all the gel.
• Prior to adding the fragment-laden gel pieces, add 500� of glassmilk.
• Once the gel is in the tube, melt at 55^o C for 5 min (flicking the tube at least every 1 and 1/2 minutes).
• Then, spin out the solution for 30", wash 2X with NEW wash, spinning 30" each time, dry the filter by spinning for 1'.
• Elute the fragment by using 10-20� of elution buffer, incubating at 37^o C for 5 min (flick the tube at least 2X in that period), spinning down, and then repeating to get any remaining DNA.
• Precipitate the DNA overnight, using 3MTris pH 7.4 or ammonium acetate as the salt. If 3M Tris is used, incubate overnight at -20^o C. If ammonium acetate is used, incubate overnight at room temp.
• Then, do 2 X 70% EtOH washes.
• Resuspend in whatever volume looks like is appropriate.

You can dialyze overnight against 2 liters of injection buffer at this point, but it's really not necessary.

This method of prepping fragments is proving to be a really reliable and clean method. I have used it on pieces as large as 18 kB (that't the reason I went to spin columns in the first place). The spin columns are simply a GeneClean kit with a filter to catch any residual glass beads. The literature that they send says that the columns are good for DNA from .2-200kB.