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[Guide to Avoiding Common Pitfalls] Always running into trouble with mouse genotyping? Read this guide and master mouse genotyping with ease!

Mouse tail genotyping is a widely used genetic experimental technique for determining the genotype of laboratory mice. Genotype identification is indispensable in the application of genetically engineered mice.

DNA Extraction of →PCR Amplification → Detection by agarose gel electrophoresis → Data processing (gel image comparison, genotype determination)

| Rapid extraction of tissue genomic DNA DNA One-step extraction experimental procedure -- Applicable to the general public PCR Authentication

Experimental method:

① Sample Collection:

10-50mg (Mice can be sampled as early as one week of age, with a tail length of approximately… 2~5mm , mouse-ear necklace 3mm x 3mm , toe 1-3 Individuals; other tissues were weighed; length and area decreased accordingly in non‑one‑week‑old specimens.

Precautions :

This method is applicable only to freshly harvested tissues; once excised, the tissue should not be left at room temperature for more than 1 Hours (samples that are not to be lyophilized should be frozen immediately) -80℃ ), ensuring the extracted genome DNA Not degraded ;)
Thoroughly clean and disinfect sampling equipment to ensure it does not contaminate the samples.

② Submit DNA Template:

Lysis buffer, protease K , anhydrous ethanol, 75%DEPC Anhydrous ethanol, DEPC Water , Set the water bath to 55℃ , the laminar flow hood was pre-irradiated with UV; centrifuge;

③ PCR ：

2×Magic Green Taq SuperMix , primer, DEPC Water, DNA Sample, PCR8 Manifold (flat cover), PCR Ritual.

④ Electrophoresis gel running ：

High-purity, low-electroosmotic agarose, nucleic acid dye, 1XTAE Solution, gel‑casting mold, comb, appropriately sized DNA marker , electrophoresis apparatus, imaging system, and other equipment required for experiments.

⑤ Experimental method:

① Add to each sample 300ul Lysis buffer +5ul PK ， 55°C Digest overnight (no less than 4h ) The lysis buffer is prepared as shown in the figure below:

1M Tris HCL (pH=8.0)	10 ml
0.5M EDTA	4 ml
1M NaCl	400ml
10%SDS	100ml
Add MilliQ to 1000 ml

② Ethanol precipitation: Join 600ul (Double volume) -20℃ Pre-cooled anhydrous ethanol; invert the tube until a flocculent precipitate is clearly visible, then centrifuge. 12000rpm，5min , discard the supernatant;

③ Add 1 ml 75% DEPC Anhydrous ethanol, centrifugation 12000rpm，5min；

④ Dry: After centrifugation, carefully aspirate the supernatant. Place the sample on a clean bench to air-dry. 30-60min Until completely dry, at which point the precipitate becomes transparent, the extracted… DNA High quality;

⑤ Join 50 µL DEPC Water: dissolve thoroughly by shaking vigorously up and down. DNA ；

⑥ Obtain the genome DNA solution 3.0 μl , run 1.5% Agarose gel electrophoresis, detection DNA Extraction quality;

⑦ Collected DNA Can be used downstream. PCR For molecular biology experiments, for long-term storage, please place in -20℃ , to prevent DNA Degradation.

⑥ Common PCR Reaction system

Needs to be set up Positive control (If available, use positive mouse samples as controls to rule out false negatives.) Wild control (Using wild-type or heterozygous mouse samples as controls) and No-template blank control (Adopted DNase Free Water Replacement template DNA , exclude PCR Systemic pollution).

⑦ Common PCR Reaction conditions

Please based on the primer Tm Set the annealing temperature; typically, set it lower than the primer’s melting temperature. Tm Value 3-5℃ That's all;
Such as primers Tm Value ≥72℃ , the annealing step can be omitted, and the subsequent extension step can be performed directly. ( Two-step method PCR) ；
If necessary, it is recommended to establish a temperature gradient to identify the optimal annealing temperature for primer–template binding.
If poor amplification specificity is observed, the annealing temperature can be appropriately increased. ;
Annealing temperature and extension time vary depending on the brand. PCR Reagents may vary; it is recommended to consult the reagent’s product insert or contact the supplier. .

⑧ PCR Product electrophoresis analysis or product sequencing analysis

Gel formulation: According to The expected size of the PCR product bands: prepare a 1%–4% agarose gel by dissolving 1–4 g of agarose in 100 mL of TAE buffer, heat in a microwave until the agarose is completely dissolved, add 10 μL of nucleic acid dye (an EB substitute), pour the gel into the electrophoresis casting tray, insert the comb, and allow it to cool before use. 02
Sample addition: Add an appropriate amount to the wells of the agarose gel. PCR products, and add an appropriate DNA marker according to the size of the target band; 03
Running gel: Electrophoresis instrument settings 120V voltage, run for 20–30 minutes; 04
Imaging: Observe and capture images using a gel imaging system. 05
Edit the collage: Genotypes were determined based on band patterns.
06, For samples that cannot be separated by conventional electrophoresis, such as… In the SNP project, PCR products were further subjected to Sanger sequencing, and genotypes were determined by comparative analysis of the sequencing chromatograms.

07, Issue a genotype identification report.

Although this task is quite basic, you really do run into problems often—it’s not just a matter of a few words. —— Rat tail DNA PCR—— It’s easily resolved. The root cause often lies in details you’ve overlooked. Below are three common issues and their solutions. ↓

⑨ Report Description

There is no standardized notation for genotypes; rather, it largely reflects the conventions of individual users.

Example: APP/PS1 Mouse Genotype Identification

Q: Besides newly introduced mice in the laboratory, what other situations require genotyping?

Before breeding pair formation

Genotyping the parental lines ensures their genotypes are accurate, prevents source‑level errors from causing offspring confusion, and enhances breeding efficiency and the reliability of experimental results.

Offspring mice

If the parental generation exhibits genotypic diversity—such as when heterozygotes are mated—the offspring must undergo genotyping. By identifying and selecting mice with the specific genotypes required for the experiment, we can ensure the study proceeds smoothly.

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