General protocol
Kit Components:
- KAPA Express Extract (1U/µl)
- KAPA Express Extract Buffer (10X)
- KAPA2G Fast Genotyping Mix with dye
Notes
- Extract PCR-ready DNA from mouse tissue in a simple, single tube protocol (15 min), without the need for hazardous chemicals or multiple washing steps.
- Use 1 µl DNA extract per 25 µl PCR. One extraction yields sufficient template for up to 100 x 25 µl PCRs if undiluted lysate is used.
- DNA extracts may be diluted in TE Buffer (1:10) for long-term storage at -20 °C.
- KAPA2G Fast Genotyping Mixes contain a novel DNA Polymerase (with or without HotStart), engineered specifically for Fast PCR, dNTPs (0.2 mM each dNTP final) and MgCl2 (1.5 mM final).
- Use annealing time of 15 sec or less.
- Use 10 sec total extension time for most amplicons <1 kb.
- Extension time can be increased to 30 sec/kb for longer or more difficult amplicons.
- Do not exceed 25 µl PCR reaction volumes.
- KAPA2G Fast Genotyping Mix with dye allows for direct loading of PCR products into agarose gels, without the addition of a DNA loading dye solution.
1. Products
| Description |
Size |
Catalog# |
| KAPA Mouse Genotyping Kit | 10 extrac + 100 PCR | KK7301 |
| 500 rxn | KK7302 | |
| KAPA HotStart Mouse Genotyping Kit | 10 extrac + 100 PCR | KK7351 |
| 500 rxn | KK7352 |
2. DNA extraction protocol
| Step |
Description |
|
| Volume of reaction | PCR Water | Up to 100 µl |
| KAPA Express Extract Enzyme 1 U/µl | 2.0 µl | |
| KAPA Express Extract Buffer 10 X | 10 µl | |
| Mouse tail or ear tissue | 2 mm² fragment | |
| Lyse | Incubate in a thermocycler for 10 min at 75 °C. During this step, cells are lysed, nucleases and proteins degraded and DNA released. |
|
| Inactivation | Incubate for 5 min at 95 °C to inactivate the thermostable KAPA Express Extract protease. | |
| Récupération de l'échantillon | 1. Vortex reaction product for 2 – 3 sec. Centrifuge at high speed for 1 min to pellet debris3. 2. Transfer DNA-containing supernatant (~70 µl) to a fresh tube4. 3. Use 1 µl of DNA extract directly in a 25 µl PCR, without quantification. 4. Dilute in TE Buffer for long-term storage at -20 °C (optional). |
|
3. DNA Amplification
| Component |
Final Concentration |
Volume in 25 µl reaction |
| PCR Water | - | up to 25.0 µl |
| KAPA2G Fast Genotyping Mix 2X | 1X | 12.5 µl |
| MgCl2 (25 mM) | 1.5 mM in Master Mix 1X | 0.5 µl |
| Forward Primer (10 µM) | 0.50 µM | 1.25 µl |
| Reverse Primer (10 µM) | 0.50 µM | 1.25 µl |
| DMSO (for amplicons with a GC content >60%) | 5.0 - 7.5% | 1.25-1.875 µl |
| DNA | - | 1 µl |
4. Cycling Parameters
| Step |
Parameters |
Nomber of cycles |
| Initial denaturation | 3 min at 95 °C | 1 |
| Denaturation | 15 sec at 95 °C | 35 |
| Annealing | 15 sec at optimal Ta (55-65°C) | |
| Extension | 10-30 sec/kb at 72°C | |
| Final extension | 0-10 min at 72°C | 1 |
5. Results
 |
| Results for five amplicons (312 – 915 bp) generated with the KAPA Mouse Genotyping Kit (1) were compared to those obtained with two commonly used methods (2 and 3). With the KAPA Mouse Genotyping Kit, DNA lysates were prepared from mouse tails with the rapid (15 min), single-tube KAPA Express Extract system. Amplification with the KAPA2G Fast HotStart Genotyping Mix with dye was completed in 45 min. In contrast, DNA lysates were prepared with a ~3.5-hour Proteinase K protocol (2) or a rapid (~2.5 h) alkaline lysis method (3) (top panel). In both cases, amplification was performed with wild-type Taq (1.5 h cycling protocol). Results obtained with the KAPA Mouse Genotyping Kit were equal or better (more specific) than those obtained with other methods, which (depending on the exact DNA extraction protocol used), may take at least four times as long, or up to 1 day to complete. |