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:
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.
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:
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:
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.