KAPA3G Plant PCR Kits

• Direct PCR from crude plant samples including leaf tissue and seeds
• Crude PCR from leaf tissue and seeds
• Successful amplification of CTAB-extracted DNA
• Streamlined workflows for transgenic screening
• As a solution for improving yield, sensitivity and specificity of PCRs where amplification is difficult or impossible to achieve with standard reagents

Reaction set-up

• Amount of starting template: For high quality DNA, 1 – 10 ng genomic DNA per 50 µL reaction should be sufficient for most applications. For crude samples, DNA contaminated with inhibitors and low-quality DNA, it is essential to determine the optimal template concentration per reaction in a template dilution series PCR.
• Amount of sample for direct PCR: The use of a 0.5 mm diameter sampling tool is recommended for most applications, although a 0.35 mm diameter sampling tool may yield higher quality results with problematic species/samples. Adding a crude sample into a PCR reaction together with purified DNA will give an indication of how inhibitory the sample is and whether the amount of crude sample in the reaction should be reduced.
• Amount of sample for crude PCR: 1 µL of crude leaf or seed preparation, as per the directions in the Technical Data Sheet and the KAPA3G Plant PCR Application Note.
• Enzyme amount: One unit of KAPA3G Plant DNA Polymerase per 50 µL reaction (or proportionally less for smaller reaction volumes) should be suitable for most applications. For PCR from difficult crude samples that fail with a 1 U/50 µL reaction, increase the amount of enzyme. Conversely, results may possibly be improved by reducing the amount of enzyme when non-specific amplification and/or smearing occurs.
• Primer concentration: The primer concentrations in the final reaction are 0.3 µM for each primer. If non-specific amplicons or high molecular weight smears are present, the primer concentration can be reduced; however, the final concentration should not be lower than 0.1 µM.
• Magnesium concentration: The KAPA3G Plant PCR Kit is supplied with a 2X buffer that includes MgCl₂ at a 1X concentration of 1.5 mM, which is sufficient for purified DNA. In general, a final MgCl₂ concentration of 2.0 mM is recommended for crude samples. In rare cases, smearing may occur at a final MgCl₂ concentration >1.5 mM; reduce the MgCl₂ concentration when this happens. Additional MgCl₂ (25 mM) is included in all kits for assays that require additional MgCl₂, for optimization of the final MgCl₂concentration.
• Genomic target length: Fragments in excess of 7 kb have been amplified successfully from purified DNA using the KAPA3G Plant PCR Kit. However, success with long fragments is highly dependent on template quality and primer and template characteristics.
• Reaction volumes when crude samples are used: Use 1 U (0.4 µL) KAPA3G Plant DNA Polymerase per 50 µL reaction as a first approach. Many crude sample types will work in smaller reaction volumes, containing the equivalent of 1 U enzyme/50 µL — e.g. 0.5 U enzyme in a 25 µL reaction. Species that typically require 50 µL reactions for best results are grapevine, tobacco and others with a known high polyphenol content. Species that work well in reaction volumes <50 µL include rice and maize. Efficiency of PCR when downscaling should, however, always be determined empirically.

Cycling parameters

• Denaturation time: For crude samples, an initial denaturation time of at least 10 minutes is recommended. Increasing the initial denaturation time up to 20 minutes for particularly recalcitrant templates may improve results.
• Extension time: Use 30 sec/kb as a first approach. If non-specific products longer than the desired product are observed, reduce the extension time to 20 – 25 sec/kb. Extension times that are too long may cause non-specific amplification or smearing, whereas extension times that are too short may result in low yields.
• Annealing temperature: Optimal annealing temperature is not only determined by primer and template characteristics, but also by the chemical environment (buffer, additives and sample composition). Start with the average primer Tm + 2°C as a first approach. If the results are unsatisfactory, perform an annealing temperature gradient PCR with purified DNA to determine the annealing temperature that produces the highest yield of your specific product.
• Annealing time: Use 15 seconds as a first approach. Reduce the annealing time to 10 seconds if non-specific amplification is observed, or increase it up to 30 seconds to increase yields. In general, an annealing time of 15 seconds works well for a variety of reaction volumes, thermal cyclers, and for the vast majority of primer sets. Only modify the annealing time if other optimization attempts have failed.
• PCR cycles: Use 40 cycles as a first approach, then increase or decrease the number of cycles according to the results obtained. Successful PCR with crude samples generally requires 40 – 50 cycles.

• Increase the annealing temperature or determine the optimal annealing temperature in an annealing temperature gradient PCR.
• Optimize the MgCl₂ concentration.
• Reduce the number of cycles.
• Reduce the extension time to 15 sec/kb per cycle for amplicons ≤1 kb and 20 – 25 sec/kb per cycle for 1 – 5 kb amplicons.
• Reduce the amount of template in the reaction. For high quality DNA, 1 – 10 ng genomic DNA per 50 µL reaction should be sufficient for most applications.
• Reduce the primer concentration, but do not add less than 0.1 µM of each primer.
• Reduce the annealing time to 10 sec/cycle.
• Reduce the amount of enzyme per reaction.
• Redesign primers to eliminate inter- or intra-primer interactions or improve specificity.

An extension time of 30 sec/kb should be suitable for most PCR assays, and may be reduced to 20 – 25 sec/kb if it is necessary to reduce non-specific amplification.

One unit of KAPA3G Plant DNA Polymerase per 50 µL reaction (or proportionally less for smaller reaction volumes) should be suitable for most applications. However, in the following cases more or less enzyme may yield better results:

• For PCR from difficult crude samples that do not work at 1 U/50 µL reaction, increase the amount of enzyme.
• Results may possibly be improved by reducing the amount of enzyme per reaction in the following cases:
  • When smearing occurs, particularly during the amplification of long fragments
  • When a high background of non-specific amplicons is obtained

Only optimize enzyme concentration after annealing temperature, cycle number and magnesium concentration have been eliminated as possible causes.

The KAPA3G Plant PCR Kit is supplied with a 2X buffer that includes MgCl₂ at a 1X concentration of 1.5 mM. Additional MgCl₂ (25 mM) is included in all kits for assays that require additional MgCl₂, or for the optimization of the final MgCl₂ concentration.

• Purify template DNA from the specific species/sample and use it to set up and optimize the basic PCR parameters (reagent concentrations and cycling parameters) and determine the sensitivity of the assay (minimum number of target copies detectable).
• Prepare a 10-fold dilution series of the experimental template, or use 0.35 mm or 0.5 mm diameter crude sample discs. Set up duplicate reactions for the template dilution series, in which one set is spiked with a known amount of the target (10 – 100 times more than the detection limit). For crude samples, do triplicate sets of reactions at different amounts of crude sample, also spiked into reactions with purified template, to assess the degree of inhibition, if any.
• The results from such a dilution series experiment will indicate if inhibitory element(s) in the experimental template may be diluted out, whilst remaining within the sensitivity range for the target amplicon, and also whether reliable amplification may be achieved from a particular crude sample type and/or size.

The recommended temperature for long-term storage of the KAPA3G Plant PCR Kit is -20°C. However, kit components may be stored at 4°C for short-term usage (up to one month).

The KAPA Plant PCR buffer has been formulated for optimal enzyme performance under a wide variety of reaction conditions and with diverse template and amplicon types; therefore, additional additives should not be required for the majority of applications. If a user wants to experiment with additives, the following strategies may be explored:

• Include additives such as PVPP, PVP40 or PEG 8000 in the reaction. Both PVP40 and PEG 8000 may be included at 1 – 3% (m/v) final concentration.
• Include glycerol at a final concentration of 2.5% to address very problematic non-specific amplification.
• The addition of 5% (v/v) final DMSO enables amplification from high-GC templates. The KAPA Plant PCR Buffer will typically still work with targets up to ~70% GC, without additives. Note that the addition of glycerol and/or DMSO may cause failure of amplification from low-GC templates
• Other PCR additives may be investigated using a systematic approach. KAPA 3G Plant DNA Polymerase typically tolerates higher concentrations of additives than wild-type Taq, but the relative advantage and optimal concentration of each additive will have to be determined empirically.

PCR products generated with the KAPA3G Plant PCR Kit have the same characteristics as PCR products generated with KAPA Long Range, and are suitable for routine downstream applications such as digestion with restriction endonucleases (RE) and sequencing. PCR products generated with the KAPA3G Plant PCR Kit are 3′-dA-tailed and may be used for TA cloning or may be blunt-ended or digested with restriction endonucleases prior to cloning. For best results, purification of PCR products using any standard column or bead-based PCR cleanup kit is recommended.

The KAPA3G Plant PCR Kit can be used for multiplex PCR, although optimization of reaction parameters is likely to be required.

Direct- and crude-sample PCR are challenging applications and it is difficult to predict which amplicons can be successfully amplified from which crude sample types. The protocol given below is a starting point for the preparation of crude templates and crude sample PCR using KAPA 3G Plant PCR Kits:

• For leaf samples, or plant sap samples spotted onto filter paper, use the 0.5 mm diameter Harris Uni-Core, or a similar sampling tool, to obtain a disc that can be added directly to a PCR reaction. Remember to also add crude sample to a reaction containing a known amount of purified DNA to gain information about the amount of inhibition caused by the crude sample. If no amplification occurs in this “spiked” reaction, then the sample is very inhibitory, and the size of the crude sample needs to be reduced even further, or a dilution protocol has to be followed. A 0.35 mm diameter Harris Uni-Cor sampling tool is also available, and may be useful for extremely inhibitory samples. Some types of samples are more suitable than others for use with such a small diameter sampling tool, such as grapevine leaves, as they have a much firmer structure and are easier to work with. Grapevine leaf discs of 0.35 mm diameter may also give better results than 0.5 mm discs, because of the very inhibitory nature of the material.
• As an alternative to plant punches, and to allow for multiple PCRs from one preparation, the extraction buffer, as described in the User Guide, may also be used.
• Sample leaf material for PCR as soon as possible after the material is procured. If plant material was frozen and thawed it is particularly important to set up the PCR as quickly as possible and start cycling immediately.
• If no amplification can be achieved with crude material directly in the reaction, or with a preparation using the extraction buffer, try diluting the extract. If diluting the extract also doesn’t work, then this sample type may not be suitable for crude sample PCR or the DNA might be degraded.
• Seed material may also be sampled with the 0.5 or 0.35 mm sampling tools, or with the extraction buffer, using a similar approach as discussed above.

Thus far, this kit has been successfully used for amplification from dried leaves of Arabidopsis, bean, lemon, maize, rice, sugarcane, tobacco, Eucalyptus, birch, rice and tomato (amongst others). Direct amplification from dried leaves will not work for all species, such as dried grapevine leaves, but should work when extracted with the extraction buffer described in the User Guide. It will also depend on the age of the leaves and the storage conditions. Amplification from dried seed material of maize, soybean, bean, canola, wheat, linseed and pea (amongst others) has been achieved. It is recommended that for initial testing, seed material is used directly in the reaction, but that reactions using different volumes of a crude extraction preparation as template are also run. This would also depend on the testing requirements—a larger number of tests required from one seed might indicate use of the extraction buffer as the method of choice, whereas once-off testing might indicate direct PCR.

• The KAPA3G Plant PCR Kit works very well for amplifying from CTAB-extracted DNA.
• The KAPA3G Plant PCR Kit may also be used for amplifying DNA isolated with commercial “crude” DNA extraction methods, such as the Sigma Extract-N-Amp method.