Preparation of DNA for Gene Targeting

Linearize 100-200 micrograms of the targeting vector using whatever enzymes are required for the targeting strategy. Precipitate the DNA and wash the pellet with 70% ethanol. Work under sterile conditions from the 70% ethanol wash stage onwards. Air-dry the pellet and resuspend it in sterile TE or sterile PBS at approximately 1 microgram per microliter. Run 0.2 micrograms of the DNA on an analytical agarose gel with appropriate standards to confirm that it has the required form and is at the right concentration. Provide this DNA, a picture of the gel and ideally a rudimentary map of the construct noting drug resistance elements, to the core facility. Ensure that you have made clear the nature of the drug selection strategy and the type of embryonic stem cells to be used.

Note that many gene targeting experiments stall at the screening stage because insufficient prior effort has been invested in ensuring that the screening strategy is robust and effective. If using a southern blot strategy, it is essential to test the probes being used in advance to make certain that they hybridize to bands of the expected size in the absence of excessive background hybridization. A Southern blot strategy usually requires that the probe and the targeting vector should not share sequence in common. The wild-type band detected by the probe should be clearly separated from the mutant band, and the choice of enzyme used for digestion of the genomic DNA should be based on cost and the efficiency of cutting DNA that is not highly purified. Differences of 10 vs. 11Kb or 20 vs. 18Kb may be difficult to detect when working with small amounts of DNA from hundreds of clones. A PCR strategy should be tested using appropriate control plasmids and ideally should involve small amplified regions and highly optimized reactions.