The difficulty with a single residue substitution is to genotyping animals efficiently despite the difference in single residue. Special use of a restriction enzyme adds only a single, rapid, and low-cost step to typical PCR-based genotyping. This protocol can be applied to any other mouse model with a point of mutation if the target sequence is recognized by a restriction enzyme, which is usually the case.
Cut one to three millimeters of the tail tip with clean scissors and transfer it to a 0.2-milliliter eight-strip PCR tube. After containing the tail sample as described in the manuscript, store it at T-minus 20 degrees Celsius until the extraction, up to about a week. Add 100 microliters of tissue lysis solution per tube and mix well, followed by spin-down using a tabletop minicentrifuge at 2, 200 times G for 10 seconds at room temperature.
Ensure the tail samples are submerged in the solution. Set the tubes onto a thermal cycler set by programming the parameters for tissue lysis, inactivation, and holding. Store the tissue lysate at four degrees Celsius for up to a week if PCR cannot be performed immediately.
Prepare a PCR solution containing five microliters of nuclease-free water, 0.75 microliters of 10-micromolar forward and reverse primers, and 7.5 microliters of PCR master mix per sample into a new PCR tube. Add one microliter of the previously prepared tissue lysate to the PCR solution. Mix the PCR solution well and spin down with a tabletop minicentrifuge at 2, 200 times G for 10 seconds at room temperature.
Set the tubes onto a programmed thermal cycler. Once the reaction is completed, store the PCR products at four degrees Celsius for one to two months or approximately one year at minus 20 degrees Celsius. Prepare the enzyme solution containing seven microliters of nuclease-free water, two microliters of CutSmart buffer at 10X strength, and one microliter of NlaIII restriction enzyme.
If there are several samples, multiply each content to make the mixture and aliquot 10 microliters per tube. Add 10 microliters of PCR product previously obtained to the enzyme solution per tube. Mix well and spin down with the tabletop minicentrifuge as demonstrated previously.
Set the tubes onto a programmed thermal cycler or heat block. Store the product after incubation under cold conditions. Add 0.75 grams of agarose in 50 milliliters of single-strength TAE buffer.
Mix and heat the agarose in the microwave until it dissolves completely. After cooling it down, add five microliters of DNA stain and gently mix. Pour 25 milliliters of the agarose gel into the gel mold and allow the gel to solidify.
Load 10 microliters of digested PCR product to the well. Run the gel with 100 volts for 35 minutes. Image the agarose gel under ultraviolet light.
The agarose electrophoresis performed using the digested PCR product resulted in several bands of different sizes in each genotype, with two bands in wild type, three in heterozygous, and one in homozygous, respectively. Test injection with blastocyst analysis resulted in a 76.9%success rate on injecting 50 nanograms per microliter of the donor oligos, and a 25%success rate on injecting 25 nanograms per microliter of the same donor oligos, respectively. Finally, injecting 50 nanograms per microliter of the donor oligos with ethanol precipitation obtained founder animals with a 50%success rate.
Purification of oligo donors by ethanol precipitation lowered the toxicity while retaining high genome editing efficiency. Careful handling and in adding a low amount of reagents is very important. Ensure they are correctly added and are mixed well.
