RRC-TPS: Preparing DNA for Microinjection: Protocol
Vector sequences can interfere with transgene expression; therefore, the plasmid backbone must be removed from your construct to release the insert (gene fragment) that you wish to have injected. This fragment must have a promoter, a start codon, a cDNA/genomic DNA/or reporter gene that you want to express, a stop codon, and transcriptional stop signal with a poly A addition sequence. Expression of cDNAs in transgenic mice may further be improved by inclusion of a heterologous intron (e.g.,rabbit β-globin intron or SV40 intron). There is no limit to the length of the fragment but the longer it is the greater the care one should take to make sure it is not sheared during isolation. BACs may also be injected (contact TPS Director).
1. Cut the plasmid containing your transgene with the appropriate enzyme(s) to release the insert. Run a minigel to check the digest. If the digest is okay, run the cut DNA in a 0.8% - 1.0% agarose gel in TAE. Use a preparative comb, or several wells of a standard comb. Stain briefly with Ethidium Bromide. Staining the gel after running helps to minimize exposure to light/EtBr, but we have included ethidium bromide in preparative gels numerous times with no problem. Cut the band of interest under long-wave U.V. light (work very quickly to limit exposure of DNA to U.V.).
2. Extract the transgene fragment from the agarose band using any of a number of commercial kits. “GeneClean” (MP Biomedicals) is a good option. We prefer the “GeneClean Spin Kit” to the original GeneClean because the Spin Kit is less likely to shear the DNA and it does not contaminate the sample with glass (silica) beads, which will clog the microinjection needles and make it impossible to inject your sample. You may also use Qiagen Kits (Qiaex II and QIAquick) but the former kit may also introduce loose silica beads, while the latter kit is not suitable for fragments longer than 10kb. If you use loose-bead methods, please take time to clear your sample of beads - this can be done by successive centrifugation steps, with careful removal of the supernatant (leaving some behind so as not to pick up the beads). Make sure to follow any product-specific instructions related to preparing DNA for microinjection.
3. Ethanol-precipitate the DNA: add 1/10 volume 2.5-3.0M NaAc (pH 5.2), then 2 volumes ice cold EtOH. Place at -20C O/N.
4. Spin and wash the DNA pellet three times with 1-1.5ml 70% ethanol each time. Remove excess EtOH from the final wash with a thin pipet (for example, a Pasteur pipet drawn-out in a flame), and air-dry the pellet for 30 min or dry under vacuum. The importance of the washing and drying steps cannot be overemphasized, as both residual salt and ethanol are lethal to a developing embryo.
5. Resuspend the pellet in a small volume of injection buffer (10mM Tris, pH7.4; 0.25mM EDTA - please come get aliquot of injection buffer from the TPS) and determine the final concentration of DNA, along with the 260/280 ratio. Submit your sample, at 50-100ng/µl (about 25-50µl). We will dilute the DNA to the appropriate concentration for injection. Also submit a picture of the isolated fragment run in a gel.
This protocol works well for us. Other facilities report using other protocols (e.g., centrifugation on CsCl gradients followed by extensive dialysis; or for large fragments electroelution followed by purification using the GeneClean Turbo Kit) with great success. If you choose to use an alternative method please provide us the protocol when you submit your sample. The DNA should be as clean as possible, free of salt and other contaminants that could result in decreased embryo survival, and prepared using a method that will not shear the fragment.