KAPA Mouse Genotyping Kit FAQs

What are the recommended applications for the KAPA Mouse Genotyping Kit?
KAPA Mouse Genotyping Kits are ideally suited for the rapid extraction and amplification of DNA from mouse tail, ear and toe tissue. The kits are also suitable for the extraction and amplification of DNA from other animal tissue, but protocol optimization may be required.

What are the major benefits of using KAPA Mouse Genotyping Kits?
KAPA Mouse Genotyping Kits contain everything needed for the rapid extraction and amplification of DNA from mouse tissue:

• KAPA Express Extract allows for a 15-minute extraction using no organic solvents and requiring no pH neutralization or centrifugation.
• KAPA2G Fast Genotyping Mix results in a 45-minute amplification using a novel DNA polymerase engineered for high processivity and high speed.
• Available in a convenient master mix format complete with loading dye, in both hot start and non-hot start formulations.

What is included in the KAPA Mouse Genotyping Kits?
The KAPA Mouse Genotyping Kits include KAPA Express Extract, a novel thermostable protease and buffer system that allows for the extraction of PCR-ready DNA from mouse tissue in as little as 15 minutes, and KAPA2G Fast Genotyping Mix with dye, which contains a DNA polymerase that has been engineered via a process of directed evolution for high processivity and extreme speed.

How much mouse sample should I put in my extraction reaction?
Approximately 2 mm³ fragment of a mouse tail or a 2 mm² punch of ear tissue should be taken into the extraction.

Did the lysis fail if tissue is still visible in the extraction reaction?
No, the tissue will not degrade completely, unlike Proteinase K digestions. This is normal, and is by no means an indication of lysis failure.

Can I quantify my extracted DNA prior to PCR?
Because of the crude nature of the extraction using Express Extract, quantification prior to PCR is not informative. Instead, rather start by using 1 µL crude extract in the PCR (preferably diluted 1:10 with TE or 10 mM Tris-HCl, pH 8.0–8.5 to remove potential inhibitors), and increase or decrease as necessary after the initial experiment.

Is centrifugation a requirement prior to PCR?
The centrifugation step is optional. If the lysate will be stored long-term, then centrifugation and transfer of the supernatant to a fresh tube prior to dilution in a buffered solution is recommended. Centrifugation of the reaction product for 1 minute at high speed is sufficient to pellet debris, and the supernatant is easily recovered. In some cases, the debris does not pellet but rather remains suspended throughout the extract. In such cases, it is recommended that the DNA-containing liquid be carefully transferred to a fresh tube for downstream use and storage.

How many PCR reactions can be performed from a single DNA extraction reaction?
One extraction yields sufficient template for up to 100×25 µL PCRs if undiluted lysate is used. Use 1 µL DNA extract per 25 µL reaction as a starting point.

What are the key areas of optimization if:

No or low yield of target fragment after PCR?

• Increase lysis at 75 °C to 15 min to improve release of DNA.
• Dilute DNA extract with TE or 10 mM Tris-HCl, pH 8.0–8.5 prior to PCR.
• Use at least 40 PCR cycles.
• Increase extension time to 20 or 30 seconds per cycle.
• Decrease annealing temperature by 2 °C–5 °C, but do not go lower than 50 °C.
• Ensure that tissue was completely immersed in the buffer during extraction.

Smeary or non-specific amplification?

• Reduce number of PCR cycles to 35 or less.
• Reduce annealing time to 15 seconds and extension time to 5–10 seconds per cycle.
• Increase annealing temperature by 2 °C–5 °C
• Prepare fresh primer stocks.

What are the recommended extension rates for the KAPA2G Fast Genotyping Mix?
10 sec/kb is recommended for most amplicons <1 kb. The extension time can be increased up to 30 sec/kb for amplicons that amplify with low yield. A final extension is only required if 3′-dA-tailing is required for fragment analysis or cloning into TA cloning vectors.

What is the optimal annealing temperature and time for KAPA2G Fast Genotyping Mix?
The optimal annealing temperature for any PCR assay depends on the specific template–primer combination. For optimal results, design primers to have an optimal annealing temperature between 55 and 60 °C. Primers with lower annealing temperatures may be used, but annealing temperatures <50 °C are not recommended. Never exceed an annealing time of 15 seconds per cycle, as this may lead to non-specific amplification and/or smearing.

What is the optimal number of cycles for KAPA2G Fast Genotyping Mix?
The optimal number of cycles depends on template concentration. Start with 35 and increase or reduce as needed.

Does magnesium need to be added to the PCR reaction with KAPA Fast Genotyping Mix?
KAPA Fast Genotyping Mix contains MgCl₂ at a 1X concentration of 1.5 mM. Additional MgCl₂ can be added if necessary for optimal results (0.5 µL of a 25 mM stock for each 0.5 mM MgCl₂ >1.5 mM).

What is the difference between KAPA2G Fast Genotyping Mix and KAPA2G Fast HotStart Genotyping Mix?
KAPA2G Fast HotStart Genotyping Mix contains KAPA2G Fast DNA Polymerase and a proprietary antibody that inactivates the enzyme until the first denaturation step. This eliminates spurious amplification products resulting from non-specific priming events during reaction setup and initiation, and increases overall reaction efficiency and sensitivity. When working with the non-hot-start mix, it is essential to work on ice during PCR setup.

What is the dye included in the KAPA2G Fast Genotyping Mix?
The dye included in the KAPA2G Fast Genotyping Mix allows for the loading of PCR products on gels directly after PCR. It contains a mixture of two dye components, which gives the mix a green color at the time of loading. This resolves into a blue and an orange component during electrophoresis. The blue band migrates with fragments of 3 – 4 kb on a 1% gel, whereas the orange band migrates with fragments <50 bp.

What are the storage recommendations for KAPA Mouse Genotyping Kits?
KAPA Mouse Genotyping Kits may be stored at 4 °C for regular, short-term use (up to 1 week). Provided that the kit has been handled carefully and not contaminated, the kit components are not expected to be compromised if left (unintentionally) at room temperature for short periods of time (up to 24 hours). Long-term storage at room temperature or 4 °C is not recommended.

How should I store my DNA extracts?
DNA extracts generated with KAPA Express Extract may be stored at -20 °C for use in multiple PCRs over a period of time. Storing in smaller aliquots is also suggested. A 1:10 dilution of the DNA extract in TE Buffer is recommended for long-term storage, to ensure that the DNA is stored in a buffered environment. However, the dilution factor may be varied between 1:1 and 1:20, depending on the downstream application and yield of DNA. For downstream applications that are sensitive to EDTA, TE may be replaced with 10 mM Tris-HCl, pH 8.0–8.5.

Can samples extracted by alternative protocols be used with the KAPA2G Fast Genotyping Mix?
Yes, KAPA2G Fast Genotyping Mix can be used with DNA extracted from mouse tissue from a variety of methods. The optimal amount of DNA extract for use in genotyping PCR should be determined by performing PCR with various dilutions of template.

Can products generated with KAPA Mouse Genotyping Kit be digested, cloned, and sequenced?
Yes, PCR products generated with KAPA Mouse Genotyping Kits have the same characteristics as PCR products generated with wild-type Taq polymerase. They may be sequenced or digested with restriction endonucleases using standard protocols. Products are 3′-dA-tailed and may be used for TA cloning, or may be blunt-ended or digested with restriction enzymes prior to cloning. For best results, purification of PCR products using any standard PCR cleanup kit is recommended.

Can the DNA extracted be used directly in a qPCR reaction?
Yes, DNA can be extracted from commonly-used mouse tissues and used as template in mouse genotyping qPCR assays with KAPA SYBR^® FAST. The sample should be centrifuged for 1 min to pellet debris after heat inactivation of the KAPA Express Extract Enzyme and the DNA-containing supernatant should be transferred to a separate tube. The recommended reaction setup and cycling protocol are as follows:

KAPA SYBR FAST reaction setup per 20 µL reaction

• PCR grade water: 8.2 µL
• KAPA SYBR FAST 2X qPCR MasterMix: 10.0 µL (1X final)
• Forward primer (10 µM): 0.4 µL (200 nM final)
• Reverse primer (10 µM): 0.4 µL (200 nM final)
• KAPA Express Extract DNA: 1.0 µL

KAPA SYBR FAST cycling protocol

• Enzyme activation: 95 °C for 3 minutes
• Denature: 95 °C for 5 seconds (40 cycles)
• Anneal/extend: 60 °C for 30 seconds (40 cycles)
• Dissociation: According to instrument guidelines