How to Perform Quick Mouse Genotyping–6 PCR Tips

Traditionally, genomic DNA is purified from mouse tissue samples for genotyping PCR. Even with fast extraction kits, the process can take 0.5–1 hour at minimum and rely on special lab equipment like centrifuges and heat blocks. Extraction reagents are also needed, some of which may call for proper disposal.

The good news is that there are direct PCR kits, allowing you to add tissue directly into PCR reactions for amplification. There’s no need for DNA purification or handling of special equipment and additional reagents (Figure 1). With a starting sample like tail snip, ear punch, or hair with follicle, direct PCR can save you significant time right from the beginning (Figure 2).

You may wonder about saving the sample for later usage. In fact, there’s an option of lysing the samples for DNA and storing it long term for your convenience (Figure 3).

We all know PCR optimization can be a pain. If you use multiple sets of primers, finding their optimal annealing temperatures is a tedious process. Worse, running PCR reactions on the same block may not be possible if their annealing temperatures differ widely. That could mean a long wait between each run.

Luckily, there’s a direct PCR master mix with a universal annealing feature. That means you can use 60°C as the annealing temperature for your primers. This will set you free from spending a lot of time having to calculate annealing temperatures and run assays on separate blocks. (Figure 4).

PCR reactions contain multiple components like the buffer, enzyme, dNTPs, MgCl₂, primers, and DNA template. That means setting up PCR experiments can be time consuming and error prone, with an additional risk of reagent contamination. All these hurdles can be avoided with a universal master mix that contains all necessary components for PCR; all you need to add are your primers and tissue sample (Figure 5).

No one likes to wait a long time for PCR to complete. Luckily, PCR reaction time can be shortened in the following three aspects, in their decreasing order of impact:

1.  Look for fast PCR enzymes like those that can synthesize DNA at 15–20 sec/kb instead of 60 sec/kb.
2. Use a fast thermal cycler in combination, such as one that can ramp up or down to the desired cycling temperatures at a fast rate (e.g., 6 sec/°C).
3. Choose “fast” plastics (when appropriate), which feature low profile for enhanced thermal conductivity and ultra-thin walls for better heat transfer.

Using “fast” enzymes, thermal cyclers, and plastics, PCR amplicons of ≤1 kb can be amplified in as little as 40 minutes (Figure 7).

Traditionally, a loading dye is added to completed PCR reactions before loading samples onto a gel for tracking and density gradient. Seemingly trivial, the process involves calculating the volumes needed, making a loading dye, pipetting out PCR samples, and finally mixing them. These steps take time, not to mention extra plastics waste created along the way. It can be a thing of the past for you with a direct PCR master mix that already includes an inert dye for direct gel loading (Figure 8).

PCR products are routinely run on agarose gels to analyze mouse genotypes. What if you can skip the traditional electrophoresis workflow of gel pouring, buffer making, gel boxes assembling, and gel staining or destaining? What if you can just load your samples and start the run?

The good news is that there’s an option to just do that! Analyze PCR of mouse genotyping in three simple steps—load, run, and analyze (Figure 9). Better yet, a camera hood is integrated in some systems for real-time viewing of the running gel. Combining with “fast” gels, you can separate 50 bp–2 kb fragments in as little as 5 min!

Equipping you with these tips, we hope you can shave off significant time in mouse genotyping and focus on your critical experiments. (Bonus benefit: Your lab mates may also thank you for not hoarding the lab’s thermal cycler.)