Mouse Genotyping protocol
Step 1: Gather Materials and Reagents
- Make sure you have all the necessary materials and reagents ready for the genotyping process. This includes the tissue samples (tail snips, ear punches, or other tissues), Extraction Reagent, Stabilization Buffer, PCR reagents, and agarose gel.
- AccuStart™ II Mouse Genotyping Kit (Cat. No. 95135-100; Size: 100 reactions; Quantabio)
Step 2: Prepare Tissue Samples
- Depending on the type of tissue samples you have (tail snips, ear punches, or other tissues), determine the appropriate volume of Extraction Reagent needed. Refer to the table provided earlier for guidance on sample sizes and Extraction Reagent volumes.
- Ensure that tissue samples are small and completely submerged in Extraction Reagent.
| Sample Type | Sample Size | Extraction Reagent Volume | Comments |
|---|---|---|---|
| Tail Snips | 2 mm | 100 μL | Fresh or frozen tail snips can be used. Use proportionally more Extraction Reagent with tail snips larger than 2 mm. Samples will appear to remain intact and will not dissolve in Extraction Reagent after heating. |
| Ear Punches | 2 mm | 50 μL | Ensure that the ear punches are completely submerged in Extraction Reagent. |
| Other Tissues | 5 mg | 100 μL | Tissue samples should be small and completely submerged in Extraction Reagent. |
Step 3: DNA Extraction
- Add the tissue samples to the appropriate volume of Extraction Reagent as calculated in Step 2.
- Heat the samples to 95°C for 30 minutes. Note that the tissue samples will not dissolve and will appear intact after incubation.
- Cool the samples to room temperature.
- Add an equal volume of Stabilization Buffer to the cooled samples. This serves as a safe stopping point.
- Extracts can be stored at 4°C for several weeks or at -20°C for several months.
Step 4: PCR Reaction Setup
- Use up to 2.5 μL of the DNA extract in a 25 μL PCR reaction. Depending on the sample size and extraction conditions, you may need to dilute the extract in water or TE buffer for optimal PCR results.
- Follow your standard PCR reaction setup protocols for amplifying the DNA regions of interest.
Step 5: Gel Electrophoresis
- Load 5 or 10 μL (or as appropriate) of the PCR products on an agarose gel.
- Perform gel electrophoresis to visualize and confirm the presence of the desired DNA fragments.
Additional Notes:
The protocol can be optimized for specific applications by adjusting incubation times or omitting the addition of Stabilization Buffer if needed.
It's recommended to use PCR tubes or multi-well plates and incubate in a thermal cycler with a heated lid to prevent sample condensation.
