Streamlined Genotyping of Mouse Tissue Using DBdirect^™ PCR Gel Mixes

Genotyping is a critical step in mouse model research, providing essential information about genetic modifications. Traditional genotyping protocols often involve complex, time-consuming DNA extraction procedures. However, recent advances have simplified the process, such as the DNA isolation-free PCR method using DBdirect™ PCR Gel Mixes. This application note presents a streamlined protocol designed for efficient genotyping of mouse tail, ear, and toe clippings without the need for traditional DNA extraction.

Key Benefits of the DNA Isolation-Free PCR Approach

The DBdirect™ system offers several advantages over traditional methods:

Time Efficiency: By eliminating the need for DNA extraction, the protocol significantly reduces preparation time, allowing researchers to process samples more quickly.

Simplified Workflow: With direct PCR from tissue samples, the handling steps are minimized, reducing complexity and the potential for human error.

High Throughput Compatibility: Ideal for high-throughput settings, the protocol allows for faster processing of large sample numbers, facilitating quicker genotyping results.

Reduced Risk of Contamination: Fewer pipetting and handling steps minimize the risk of cross-contamination between samples.

Cost Savings: The streamlined approach reduces the number of reagents and consumables needed, lowering overall experimental costs.

Step-by-Step Workflow

The following steps outline the streamlined genotyping workflow using DBdirect™ PCR Gel Mixes:

1) Sample Collection

Tissue samples are collected from the mouse tail, ear, or toe. These small tissue biopsies are typically sufficient to obtain the necessary genomic DNA for analysis.

2) Genomic DNA Extraction

Instead of performing a traditional DNA extraction, the tissue sample is incubated in DB Lysis Buffer A (DB-1281) at 95°C for 30 minutes. This step releases the genomic DNA from the tissue into solution, preparing it for direct amplification.

3) PCR Master Mix Preparation

Prepare the PCR master mix using the 2x DBdirect™ PCR Gel Mix, which contains all necessary components for PCR amplification. Researchers simply need to add primers, the DNA template from the lysed sample, and PCR-grade water.

4) PCR Amplification

A standard PCR protocol is followed, typically involving 35 cycles, which can be completed in under 45 minutes. This step efficiently amplifies the genomic regions of interest for genotyping.

5) DNA Gel Electrophoresis

Once PCR is complete, the amplified products are ready to be analyzed via gel electrophoresis. The DBdirect™ PCR Gel Mix includes dyes that facilitate run monitoring and increase buffer density, making it easy to load and visualize the PCR products on an agarose gel.

There are two available formulations of the DBdirect™ PCR Gel Mix, each tailored to specific experimental needs:

DBdirect^™ PCR Gel Mix (DB-1272): This standard mix is ideal for most genotyping applications.

DBdirect^™ PCR Gel Mix SuperSens (DB-1275): If your assay requires enhanced sensitivity, this variant provides improved detection of low-abundance DNA.

The DNA isolation-free PCR protocol using DBdirect™ PCR Gel Mixes simplifies mouse genotyping by reducing time, handling steps, and the risk of contamination. It is particularly suited for high-throughput applications, providing reliable results while saving both time and resources. Whether working with a small or large number of samples, DBdirect™ PCR Gel Mixes offer a streamlined, cost-effective solution for mouse genotyping.