The KAPA Express Extract Kit is ideally suited for the extraction of DNA from crude samples for most PCR applications, including qPCR analysis; however, residual inhibitors may result in a quenching of commonly used fluorophores during qPCR. For optimal results, KAPA2G Robust HotStart ReadyMix PCR kits are recommended for downstream PCR applications.
Depending on the sample type, a single extraction typically yields sufficient template DNA for 50 – 500 standard PCR reactions. Usually 1 – 2 μL of the extracted supernatant is used in a PCR reaction (50 – 100 PCRs). To determine how many PCR reactions can be achieved from a single extraction, prepare a serial dilution with TE or Tris pH 8.0 with a small volume of extracted sample. With each serial dilution, perform a PCR to determine the optimal dilution factor.
KAPA Express Extract Kits have been extensively tested for the extraction of DNA from a wide range of sample types. Depending on the sample type, various DNA damaging agents and/or inhibitors are likely to be present that can affect both the extraction process and the downstream PCR. Initially, optimize the PCR protocol using the following recommendations and then repeat the DNA extraction.
Depending on the sample type it is possible to omit the centrifugation step after the extraction reaction. KAPA2G Robust DNA Polymerase is recommended for downstream PCR since sample debris and inhibitors will be present at a higher concentration and will almost always result in unreliable results if other DNA polymerases such as wild-type Taq are used. Pelleting the debris will result in a more reliable PCR. It is essential to pellet blood prior to use in downstream applications.
DNA extracted using KAPA Express Extract Kits can be used directly in qPCR, however there are limitations. Primary limitations include: quenching of the fluorophore by carryover inhibitors (e.g. blood) is well known to quench SYBR fluorescence; inhibition of the qPCR reaction; and/or low concentrations of DNA from extraction protocol.
Recommendations for using KAPA Express Extract with downstream qPCR include:
Upon receipt, store the entire kit at -20°C in a constant-temperature freezer. When stored under these conditions and handled correctly, all kit components will retain full activity for at least six months from the date of receipt, or until the expiry date indicated on the kit. KAPA Express Extract Buffer and enzyme may be stored at 4°C for regular, short-term use (up to one week). Provided that it 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. Please note that reagents stored above -20°C are more prone to degradation when contaminated by the user; storage at such temperatures is therefore at the user’s own risk.
DNA extracted using the KAPA Express Extract Kit will degrade over time in 1X Express Extract Buffer and the presence of carryover inhibitors may increase the degradation process. Typically DNA extracted from “clean” samples such as buccal swabs and hair follicles are stable at 4°C or -20°C for several weeks after extraction, but this can be reduced to days depending on the sample type. A 1:5 dilution of the DNA extract in TE Buffer is recommended for long-term storage. This is done to ensure that the DNA is stored in a stable buffered environment. 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. DNA extracts stored in this manner are typically stable for at least six months. For longer-term storage (especially from samples with a high concentration of carryover inhibitors) it is recommended that the DNA be further purified using an isopropanol or ethanol precipitation to totally remove inhibitors.
Wild-type Taq Polymerase will work with DNA extracted from “clean” sample types (e.g., buccal swabs, hair follicles) but will be unreliable when amplifying more challenging samples and templates. For the highest performance amplification, the use of KAPA2G Robust HotStart ReadyMix is recommended for all sample types.
KAPA Taq DNA Polymerase, KAPA2G Fast DNA Polymerase, KAPA HiFi DNA Polymerase, and KAPA SYBR FAST qPCR Kits have all successfully been used with DNA extracted using KAPA Express Extract to amplify a range of amplicons. KAPA2G Robust DNA Polymerase is recommended for everyday use with DNA extracted with KAPA Express Extract due to improved performance in the presence of carryover inhibitors.
A xylene wash step is not required when using the KAPA Express Extract Kit. After the centrifugation step, the layer of wax is usually found on top of the buffer or on the side. The supernatant contains the DNA and is retrieved by puncturing through the wax layer on top. It is recommended to trim the excess wax around the sample using a sterile scalpel to leave only the tissue. Excess wax will not affect the extraction process, but will make it more difficult to recover the DNA.
The extraction process has been optimized for DNA. RNA will chemically degrade during the heating protocol and RNA extraction is not recommended with the KAPA Express Extract Kit.