KAPA Express Extract Kits

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.

• Rapid extraction protocol: PCR-ready DNA in 15 minutes
• Versatility: single kit optimized for a variety of sample types
• Single-tube reaction minimizes risk of contamination
• Couple with KAPA2G Robust HotStart ReadyMix for increased PCR success rates

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.

• Human tissue (FFPE samples, blood collected in EDTA tubes or on collection cards, buccal swabs, hair follicles, forensic samples)
• Animal samples (ear or tail clippings, hair follicles, blood, bone marrow, dried or fresh meat from mouse and other mammals)
• Fish tissues (fin punches, fresh tissue, cold and hot smoked canned samples, ethanol-preserved samples)
• Insects (crushed)
• Bird feathers (calamus fragments)

• Plant samples
• Degraded samples
• Samples stored for extensive periods of time
• Samples of unknown quality

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.

• Optimization of PCR:
  • KAPA2G Robust HotStart ReadyMix or KAPA2G Robust DNA Polymerase Kits are recommended for downstream PCR using KAPA Express Extract. The KAPA2G Robust DNA Polymerase has been engineered specifically for improved processivity and tolerance to common PCR inhibitors. The robustness of the KAPA2G Robust DNA Polymerase is particularly useful for increasing PCR success rates for crude extraction applications using KAPA Express Extract.
  • Some crude DNA extracts may contain high concentrations of PCR inhibitors. If the use of KAPA2G Robust DNA Polymerase fails to yield acceptable results, it may be necessary to dilute the DNA extract prior to PCR. Repeat the experiment with a positive control sample (e.g. genomic DNA purified using a column-based kit) and a 10-fold dilution series of the DNA extract (prepared in TE or 10 mM Tris-HCl, pH 8.0 – 8.5). The DNA extracts may also be “spiked” with a known concentration of the positive control sample to determine whether the inhibitors may be diluted out, whilst retaining a high enough template concentration to support amplification of the target.
  •  Reduce the annealing temperature by 2°C – 5°C, and/or increase the annealing or extension time.
  • Non-specific products, primer-dimers and/or smearing may occur as the result of RNA or degraded DNA originating from the presence of inhibitors in the sample. Modifications to the PCR cycling protocol (e.g. increasing annealing temperature, reducing annealing and/or extension time) and using a HotStart DNA polymerase may reduce non-specific amplification.
  • DNA extracted from difficult sample types can be further purified using isopropanol or ethanol precipitation to remove inhibitors.

• Optimization of the DNA extraction:
  • The lysis step at 75°C may be varied between 10 and 30 minutes. Use a longer incubation time at 75°C to maximize DNA output (for example, rat and dog tissue require an incubation of 15 – 20 minutes).
  • For certain sample types (eg. fish), decreasing the temperature of the lysis step from 75°C to 60°C improves the efficiency of the extraction. Lysis temperature can be optimized by performing a temperature gradient from 55°C – 75°C for 10 minutes followed by incubation at 95°C for 5 minutes.
  • Heat-inactivation of the KAPA Express Extract Enzyme for at least 5 minutes at 95°C is essential, as carryover enzyme will degrade the DNA polymerase during PCR.
  • The addition of certain sample types may decrease the pH below 7. DNA is rapidly degraded in low pH environments, especially at elevated temperatures during the lysis protocol. Test the pH of the extracted DNA after the lysis protocol is completed using pH paper. If the pH is <7.5 use the KAPA Express Extract Buffer at a final concentration of 1.5 – 2X. Alternatively a smaller amount of sample can be added to the initial extraction reaction.
  • Typically DNA extracts do not have to be quantified prior to use in PCR, and quantification is not recommended, however it is possible to use a NanoDrop or equivalent to get an indication of the amount of contaminating factors.

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:

• Perform a serial dilution of the extracted DNA with TE or 10 mM Tris pH 8.0. This will dilute any potential inhibitors, reducing PCR inhibition and quenching of the fluorophore. It is likely that some quenching will remain, and the DNA concentration is likely to be underestimated.
• Quenching of the fluorophore is dependent on the type used, try using a different fluorophore. KAPA SYBR DNA Polymerase has been engineered to perform optimally in stringent real-time qPCR reaction conditions, and has reduced inhibition to SYBR Green I dye and hence elevated SYBR Green I concentrations can be used.
• The DNA extracts may also be “spiked” with a known concentration of the positive control sample to determine whether the inhibitors may be diluted out. Ideally a serial dilution is prepared from the KAPA Express Extract sample and the same DNA used at one concentration of the standard curve added to all the serial dilutions. This will give a clear determination of inhibitors (increasingly delayed Ct score across serial dilutions) or no DNA in the extracted sample (same Ct score as standard, even with undiluted sample).
• Attempt different lysis protocols (see above for recommendations) to maximize DNA output.

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.