From sample to gel results in less than an hour
Transgenic mice are widely used as a model organism in basic research to study the role of genes in development, physiology, and human diseases. PCR is a common genotyping method to detect the presence or absence of the gene(s) of interest in transgenic mice.
Genotyping PCR can be time consuming, and often you want the results fast to get to downstream experiments quickly. Here, we provide six simple PCR tips to help you overcome common hurdles in mouse genotyping. They may also be applicable to genotyping of other model organisms like fruit fly, zebrafish, and Arabidopsis.
Tip #1: Skip genomic DNA extraction
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).
Figure 2. Direct PCR with a variety of mouse tissue samples. Fragments of 0.3 kb and 3.6 kb were amplified directly from tissue using Invitrogen Platinum Direct PCR Universal Master Mix. Sample sizes were according to the recommended guideline. Molecular weight marker (M): Invitrogen TrackIt 1 Kb Plus DNA Ladder.
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).
Tip #2: Spend less time optimizing PCR annealing
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).
Tip #3: Handle fewer tubes of reagents
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 master mix that contains all necessary components for PCR; all you need to add are your primers and tissue sample (Figure 5).
Bonus tip: Depending on the genes you are detecting, you may be able to include multiple primer sets in a single reaction (i.e., multiplex PCR). In the example shown in Figure 6, up to five targets were successfully detected using a single reaction. This can save you significant time, not having to set up multiple reactions. Note though that you may need proper primer designs and optimization in the beginning for multiplexing to work.
Tip #4: Finish PCR in minutes, not hours
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:
- Look for fast PCR enzymes like those that can synthesize DNA at 15–20 sec/kb instead of 60 sec/kb.
- 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).
- 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).
Tip #5: Load PCR reactions directly to gels
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).
Figure 8. Direct gel loading simplifies PCR workflow. The green PCR master mix of Platinum enzymes oﬀers the convenience of direct gel loading of PCR products, eliminating tedious steps of dye addition to samples and helping to reduce pipetting errors.
Tip #6: Complete electrophoresis steps in <15 min
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!
Figure 9. Three simple steps for analysis of genotyping PCR. With an integrated electrophoresis system of bufferless precast agarose gels, PCR products can be loaded, separated, and analyzed in less than 15 minutes.
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.)
Six PCR tips for quick mouse genotyping
For Research Use Only. Not for use in diagnostic procedures.