Product Description

Amplification of plant-derived DNA is a challenging application due to the diversity of plant tissue types and the potent PCR inhibitors contained within the tissue. The KAPA3G Plant PCR Kit is optimized for the successful amplification of DNA from crude plant samples, DNA containing carry-over inhibitors from crude extraction methods, as well as purified DNA.

The KAPA3G Plant PCR Kit contains a novel DNA polymerase, engineered via a process of molecular evolution, for improved tolerance to common plant-derived PCR inhibitors such as polyphenolics and polysaccharides. The unique characteristics of the enzyme result in robust amplification across a wide range of plant sample types, amplicon lengths, and crude extraction methods. The kit contains 4 separate components: 1) KAPA Plant PCR Buffer (2X) is a ready-to-use cocktail containing all components except DNA polymerase, primers, and template. This 2X buffer contains 3 mM MgCl2; 2) KAPA3G Plant DNA Polymerase is a blend of an engineered Taq-based (Type A) DNA polymerase and a modified archaeal (Type B) DNA polymerase. This enzyme blend is combined with proprietary antibodies that inactivate the enzymes prior to the first denaturation step; 3) KAPA Plant PCR Enhancer is supplied as an optional additive to improve PCR performance for difficult samples and assays where MgCl2 titration fails to improve results; 4) Additional MgCl2 (25 mM) is supplied for assays that require MgCl2 optimization.

DNA fragments generated with the KAPA3G Plant PCR Kits are A-tailed and suitable for use with TA cloning vectors.

Product Applications

The KAPA3G Plant PCR Kit is designed for the amplification of DNA fragments ≤5 kb in length from a range of plant samples including:

• Crude extractions of plant DNA containing carry-over inhibitors
• Directly from leaf discs, seed samples, and other plant tissue samples
• Samples containing significant concentrations of plant-derived compounds

KAPA3G Plant PCR Kits contain a novel DNA polymerase engineered via a process of molecular evolution for improve tolerance to common plant-derived inhibitors.

Direct PCR from a variety of plant species and tissue types

The KAPA3G Plant PCR Kit is capable of amplifying DNA fragments from a variety of templates, including purified DNA (+), leaf discs (L) or seeds (S). Plant genomic DNA was purified from all species using a commercial DNA purification kit. A Harris Uni-CoreTM sampling tool (0.5 mm diameter) was used to sample leaves (all species) or seeds (all species except tobacco and Arabidopsis; for these one crushed seed was used per reaction). PCRs (50 μL) contained the crude sample or 1-10 ng purified DNA (depending on the species), and 40 cycles were performed in all cases. Targets ranged between 500 and 900 bp, and reaction products were analyzed in a 1% agarose gel. KAPA Express DNA Ladder (100, 200, 400, 800, 1600, 4000, 8000 bp) was used as a MW marker.

Streamline workflows and improve the reproducibility of results

Direct PCR using the KAPA3G Plant PCR Kit outperforms CTAB extraction and standard PCR using wild-type Taq, in significantly shorter turnaround times. CTAB-extracted DNA or leaf discs were used as templates for the amplification of targets from maize (860 bp) and tobacco (735 bp), using the KAPA3G Plant PCR Kit (top panel) or wild-type Taq (bottom panel). For each species, genomic DNA was purified in triplicate using a common CTAB extraction method. Crude material was sampled in triplicate using a 0.5 mm diameter Harris Uni-CoreTM sampling tool. CTAB-extracted DNA and crude samples were used as templates in 50 μL PCRs, with 40 cycles of amplification. Direct PCR with the KAPA3G Plant PCR Kit was completed in 45 min. In contrast, the CTAB extraction protocol required ~2 h, and the amplification with wild-type Taq 1.5 h to complete. The KAPA3G Plant PCR Kit outperformed wild-type Taq when using both CTAB- extracted DNA and crude sample. Reaction products were analyzed in a 1% agarose gel. KAPA Express Ladder (100, 200, 400, 800, 1600, 4000, 8000 bp) was used as a MW marker.

Evolved DNA polymerase enables higher performance PCR direct from plant tissue

The KAPA3G Plant PCR Kit outperforms competitor kits across a broad range of plant species, with both crude samples and purified DNA. For each species, genomic DNA was purified using a commercial DNA purification kit. Crude leaf material was sampled using a 0.5 mm diameter Harris Uni-CoreTM sampling tool. Purified DNA (1-10ng per reaction, depending on species) and crude samples were used as templates in 50 μL PCRs, with 40 cycles of amplification. Reaction setup and cycling were performed according to each manufacturers’ recommended protocol. Targets ranged between 500 bp and 900 bp and reaction products were analyzed in a 1% agarose gel. KAPA Express DNA Ladder (100, 200, 400, 800, 1600, 4000, 8000 bp) was used as a MW marker.

Successful amplification of long targets from crude samples

The KAPA3G Plant PCR Kit is capable of amplifying long targets from crude samples and purified DNA with equal efficiency. Targets of different lengths (297 bp and 4100 bp from tobacco, 640 bp from tomato, 1221 bp from grapevine, and 1448 and 2249 from potato) were amplified from purified DNA (+) or leaf discs (L) using the KAPA3G Plant PCR Kit. For each species, genomic DNA was purified using a commercial DNA purification kit. Crude material was sampled using a 0.5 mm diameter Harris Uni-CoreTM sampling tool. Purified DNA (1-10 ng per reaction, depending on species) and crude samples were used as templates in 50 μL PCRs, with 40 cycles of amplification. Reaction products were analyzed on a 1% agarose gel. KAPA Express DNA Ladder (100, 200, 400, 800, 1600, 4000, 8000 bp) was used as a MW marker.

License Information

Licensed under U.S. Patent nos. 5,338,671, 5,587,287, 5,436,146 (claims 6-16) and corresponding patents in other countries.

Document	Type	Download
KAPA3G Plant PCR Kit MSDS	Material Safety Data Sheet
KAPA3G Plant PCR Kit Brochure	Product Brochure
KAPA3G Plant PCR Kit TDS	Technical Data Sheet

 

Frequently Asked Questions

1. What are the recommended applications for KAPA3G Plant PCR Kits? The KAPA3G Plant PCR Kit is highly recommended for PCR amplification from all plant-derived DNA samples or plant crude samples. The robust performance of the enzyme allows for higher success rates and faster time-to-results than standard reagents and/or protocols. It is particularly suited for the following applications:

• Direct PCR from crude plant samples including 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

 

2. What are the most common causes of poor results (non-specific amplification, smearing or reaction failure) with the KAPA3G Plant PCR Kit?

 

• Too much crude sample in the reaction. The use of a 0.5 mm diameter sampling tool is recommended for most applications, although a 0.35 mm diameter sampling tool may give better 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.
• Suboptimal 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 specific product.
• Too few PCR cycles. Use 40 PCR cycles as a first approach. Then increase or decrease the number of cycles according to results obtained. Successful PCR with many crude samples requires 40 - 50 cycles.
• Initial denaturation time is too short. For crude samples, an initial denaturaiton time of at least 10 min is recommended. Increasing the initial denaturation time up to 20 min may improve results.
• Suboptimal 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.
• Too much 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, determine the optimal template concentration per reaction in a template dilution series PCR.
• Suboptimal amount of enzyme and/or inappropriate reaction volume 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, however, 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. A few crude sample types may require more than 1 U enzyme in a 50 µl reaction for reliable results.
• Suboptimal magnesium concentration. The standard 1.5 mM final MgCl2 concentration 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.
• Suboptimal annealing time. Use 15 sec as a first approach. Reduce the annealing time to 10 sec if non-specific amplification is observed, or increase it up to 30 sec to increase yields. In general, an annealing time of 15 s works well for a variety of reaction volumes, thermocyclers and for the vast majority of primer sets. Only modify the annealing time if other optimization attempts have failed.
• Target is too long. 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.

 

3. What should I do if PCR products generated with the KAPA3G Plant PCR Kit contain a high background of non-specific amplicons or high molecular weight smears? The high specific activity and processivity of KAPA3G Plant DNA Polymerase may sometimes result in a higher background of small or large non-specific amplicons or high-molecular weight smears when compared to products obtained with wild-type Taq (or hot start formulations thereof) under similar conditions. Since KAPA3G Plant PCR Kits contain a third-generation enzyme, some optimization may be required when it is used in assays originally designed for wild-type Taq. To improve the yield of the specific amplicon, one or more of the following may be done:

 

• 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 not lower than 0.1 µM of each primer.
• Reduce the annealing time to 10 sec per cycle. Reduce the amount of enzyme per reaction.
• Redesign primers to eliminate inter- or intra-primer interactions or improve specificity.

 

4. Should I always use 1 unit of KAPA3G Plant DNA Polymerase per 50 μl reaction? 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 some cases more or less enzyme may yield better results:

 

• For PCR from difficult crude samples that do not work at 1 unit/50 µl reaction, increase the amount of enzyme. Only increase enzyme concentration after annealing temperature, cycle number and magnesium concentration have been eliminated as possible causes of the failed reaction/s. Always include appropriate positive and negative control reactions.
• Results may possibly be improved by reducing the amount of enzyme per reaction in the following cases: 1. when smearing occurs, particularly during the amplification of long fragments, or 2. when a high background of non-specific amplicons is obtained.  

 

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

 

5. What is the recommended extension time for KAPA3G Plant DNA Polymerase? 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.

 

6. Do I have to include MgCl2 in my reaction setup? 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 optimization of the final MgCl2 concentration.

 

7. Can I store KAPA Plant PCR PCR Kits at room temperature or at 4 °C? 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 a month). Don’t worry if you’ve left any component of the kit on your bench overnight or over the weekend – it will still work fine (but don’t make a habit of it!).

 

8. When and how should I use KAPA Plant PCR Enhancer in my PCR? KAPA Plant PCR Enhancer may be used at final concentrations of 0.2X – 1X if all other optimizations have failed. It is supplied as a 100X solution.

 

9. Can I use other PCR additives with the KAPA3G Plant PCR Kit? 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, and additional additives should not be required for the majority of applications. During R & D, amplification was achieved without the use of additives with every single primer set tested on a wide variety of templates. Additives may, however, improve results in some instances. If a user wants to experiment with additives, the following strategies may be explored:

 

• Include additives such as PVP40 or PEG 8000 in the reaction. Always purchase the highest grade of any additive, and be aware of lot-to-lot variation. 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.
• For some amplifications, the addition of 2.5 % (v/v) final glycerol will increase yields. Note that the addition of glycerol and/or DMSO may cause failure of amplifications from low-GC templates.
• 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.
• 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.

 

10. How do I prepare templates for crude sample PCR using KAPA3G Plant PCR Kits? Crude sample PCR is a challenging application 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-CoreTM , or a similar sampling tool, to obtain a disc that can be added directly to a PCR reaction. Include appropriate positive and negative control reactions. Remember to also add crude sample to a reaction containing a known amount of purified DNA, in order to gauge 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-CoreTM 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. Grapevine leaves, for example, are much more suited for use with the 0.35 mm tool than e.g. potato leaves, since grapevine leaves 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.
• 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 straight away.
• If no amplification can be achieved with crude material directly in the reaction, the next approach is to crush a small amount of the sample in dilution buffer, as described in the TDS, and try a range of volumes of this extract for PCR. If this still does not work, try diluting the extract further. If diluting the extract further also doesn’t work, then this sample type may just not be suitable for crude sample PCR, or the DNA might be degraded and will never amplify.
• Seed material may also be sampled with the 0.5 or 0.35 mm sampling tools, using a similar approach as discussed above.
• Some types of seed material (wheat seed in particular) may have to be treated with a lysis reagent, such as KAPA Express Extract, in order to be used successfully for PCR.

 

11. Can I use the KAPA 3G Plant Kit with dried plant material? Thus far, the kit has been successfully used for amplification from dried leaves of Arabidopsis, bean, lemon, maize, rice, sugarcane, tobacco and tomato. Amplification from dried leaves will not work for all species – for example, amplification from dried grapevine leaves is usually very problematic. It will also depend on the age of the leaves and the storage conditions. Amplification from dried seed material of maize, soybean, bean and pea has been achieved (without the use of ExpressExtract). It is recommended that for initial testing, seed material is used directly in the reaction, but that reactions using different volumes of an ExpressExtract preparation as template, are also run.

 

12. Can I use the KAPA3G Plant PCR Kit with CTAB-extracted DNA, or with DNA samples in third-party buffers?

 

• 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-AmpTM method.

13. Can I use the KAPA3G Plant PCR Kit for Multiplex PCR? The KAPA3G Plant PCR Kit can be used for Multiplex PCR. Optimization of reaction parameters (e.g. MgCl2 concentration, relative concentration of each primer pair, enzyme and template concentrations, annealing temperature, extension time and number of cycles) is likely to be required.

 

14. How can I go about optimizing a PCR assay to be used routinely with DNA containing inhibitors or crude samples as template?

 

• 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 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.

15. Can PCR products generated with the KAPA Plant PCR be digested or sequenced? PCR products generated with the KAPA3G Plant PCR Kit have the same characteristics as PCR products generated with KAPA LongRange, and are suitable for routine downstream applications such as digestion with restriction endonucleases and sequencing. For best results, purification of PCR products using any standard PCR cleanup kit is recommended prior to RE digestion or sequencing.

 

16. Can PCR products generated with the KAPA3G Plant PCR Kit be cloned? 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 PCR cleanup kit is recommended prior to cloning.