The KAPA3G Plant PCR Kit is based on a novel, third-generation (3G) DNA polymerase, engineered via molecular evolution for improved tolerance to common plant-derived PCR inhibitors such as polyphenolics and polysaccharides. Kits are optimized for fast and efficient amplification of plant DNA from crude samples, DNA containing carry-over inhibitors from crude extraction methods, and purified DNA.
Key features of the KAPA3G Plant PCR Kit include:
• Fast PCR direct from plant tissues such as leaf discs, seeds and crude plant extracts.
• Streamlined workflows for transgenic screening.
• Improved PCR success rates and reproducibility.
• Efficient amplification of long and difficult targets from all sample types.
KK7251 | KAPA3G Plant PCR Kit (250 x 50 µL reactions) | For 250 x 50 µL reactions. Each kit contains 250 U KAPA3G Plant DNA Polymerase (2.5 U/µL), 6.25 mL KAPA Plant PCR Buffer (2X) with dNTPs, 125 µL KAPA Plant PCR Enhancer (100X), and extra 1.6 mL MgCl2. |
KK7252 | KAPA3G Plant PCR Kit (500 x 50 µL reactions) | For 500 x 50 µL reactions. Each kit contains 2 x 250 U KAPA3G Plant DNA Polymerase (2.5 U/µL), 2 x 6.25 mL KAPA Plant PCR Buffer (2X) with dNTPs, 2 x 125 µL KAPA Plant PCR Enhancer (100X), and extra 1 x 1.6 mL MgCl2. |
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.
The KAPA3G Plant PCR Kit is designed for the amplification of DNA fragments ≤5 kb in length from a range of plant samples including:
Targets of different lengths (297 bp and 4100 bp from tobacco, 640 bp from tomato, 1221 bp from grapevine, and 1448 bp and 2249 bp 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-Core™ 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.
Plant genomic DNA was purified from all species using a commercial DNA purification kit. A Harris Uni-Core™ 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.
Direct PCR using the KAPA3G Plant PCR Kit outperforms CTAB extraction and standard PCR using wild-type Taq, in significantly shorter turnaround times. 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.
Crude sample PCR with the Tab c/d primer pair was performed on leaves and/or seeds from 8 plants, with 1 μL of crude extract prepared without heat treatment (top) or 1 μL of crude extract prepared with heat treatment (bottom). Reactions were set up as described in the KAPA3G Plant PCR Kit TDS, and 45 cycles of PCR performed with annealing at 55 °C, and 20 sec extension at 72 °C per cycle. KAPA Express DNA Ladder (100, 200, 400, 800, 1600, 4000, 8000 bp) was used as a MW marker. 1: Eucalyptus; 2: Grapevine; 3: Wheat leaf; 4: Wheat seed; 5: Canola leaf; 6: Canola seed; 7: Rice seed; 8: Barley seed; 9: Corn kernel; 10: Cotton seed; 11: Cotton leaf.
Licensed under U.S. Patent nos. 5,338,671, 5,587,287, 5,436,146 (claims 6-16) and corresponding patents in other countries.