Intact Genomics FastAmp® Plant Direct PCR kit is an easy and robust method to amplify DNA fragments from plant tissues with high specificity and high sensitivity without the need of complicated DNA purification steps. The advanced formulation of this kit also allows fast PCR cycling conditions without compromising PCR sensitivity, specificity and yield. The FastAmp® Plant Direct PCR kit is an ideal and powerful tool for high-throughput genotyping, DNA amplification, and plant genome analysis.
FastAmp® Plant Direct PCR Kit is suitable for amplification of DNA directly from plant samples without purifying DNA. This kit is based on specially engineered Taq DNA polymerase, proprietary buffer system, dNTP, MgCl2 ,PCR facilitators and dye mix which makes it extremely robust and tolerant of plant PCR inhibitors such as complex polysaccharides, polyphenols and others. This kit has been tested with leaves and seeds from a wide variety of plant species. FastAmp® Plant Direct PCR Kit includes a complete set of optimized reagents and detailed protocols making it an ideal choice for amplification of plant DNA without DNA purification.
- Direct PCR- no need to purify DNA
- Specially engineered Taq DNA polymerase with highest sensitivity and specificity
- Extremely short PCR protocol times
- Master mix format with premixed gel loading dye to reduce cross-contamination and sample handling
- Useful for high GC (or non-high GC) DNA amplification
- Transgene detection
- Knockout analysis
- FastAmp® plant direct PCR master mix (2x)
- FastAmp™ plant direct Extraction Buffer
- Dilution buffer
- Control primer mix (25 µM each)
- Nuclease- free water
FastAmp™ Plant Direct PCR Kit is the Best!
A variety of plant tissues (leaves, roots, seeds) were tested from several plants using different plant specific primers for which annealing temperature and extension time were optimized separately, and also using universal (control) primers which amplify a highly conserved (297bp) chloroplast DNA fragment. Amplification was carried out directly from maize, soybean, rice, wheat, grapes, tulip, and strawberry leaf punches with different primers using Intact Genomics FastAmpTM Plant direct PCR kit and a leading commercial direct plant PCR kit (Phire plant direct PCR kit) compared side by side. FastAmpTM Plant direct PCR kit from Intact Genomics successfully amplified specific PCR products and produced results which were at par or better than the leading competitor.4615 4612<
General Guidelines before start
A. Sample handling
A leaf punch (0.5 to 1.2 mm) can be obtained by placing the leaf puncher (Uni-Core punch by GE Healthcare, Product # WB100028, or any other commercial sources) in perpendicular position over the expanded leaf and rotating it. The tip of puncher need to place inside PCR tube and expunge to drop the sample. It is critical that plant tissue materials/leaf punch is completely inside the PCR solution in tube. It is very likely that leaf punch sticks to the side of PCR tube and sometimes does not go at the bottom of tube, therefore, use a 10 µl pipet tip to manually drive the leaf punch to the bottom of PCR tube. This can be done right before capping the PCR tubes and starting the thermocycling program. For reusing the puncher, it is very important to clean the cutting edge properly with 70% ethanol to prevent cross-contamination between samples.
For seed amplification, first grind the seed and place it into 100 µl of proprietary dilution buffer. Briefly mix the tube and incubate at 95 ºC for 5-10 min. Spin down and use 1-2 µl of the supernatant as a template for a 20 µl PCR reaction.
B. Control reaction
It is recommended that universal control primers provided with this kit to be used as a positive control in all plant direct PCR reactions. Control primers are supplied as a mix of primers in H2O that amplify a 297 bp fragment of a highly conserved region of chloroplast DNA. Optimum annealing temperature for control primers is 58 ºC. As a template, use the same plant material as in the actual experiment. If the PCR using control primer mix does not work, then the plant sample may not be suitable for direct PCR. When optimizing the PCR reactions, it is recommended to perform a positive control with purified DNA to ensure that the PCR conditions are optimal.
C. PCR conditions
An initial denaturation of 5 minutes at 95°C is sufficient for most amplicons. Longer denaturation times can be used (up to 8 minutes) for difficult templates.
During thermocycling, the denaturation step should be kept to a minimum. Typically, a 20–30 second denaturation at 95°C is recommended for most templates.
Optimize the annealing temperatures for the target gene specific amplification by keeping annealing temperature at least 5 ºC below Tm values. Typically, use a 10–30 second annealing step. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.
The recommended extension temperature is 72°C. Extension times are generally 1 minute per kb for complex, genomic samples, but can be reduced to 30 seconds per kb for simple templates. When amplifying products >2 kb, it is often helpful to increase the extension time.
A final extension of 5 minutes at 72°C is recommended.
Generally, 35–40 cycles yield sufficient product.
Forward and reverse primers are generally used at the final concentration of 0.1-0.6 µM each. Too high a primer concentration can reduce the specificity of priming, resulting in non-specific products.
The PCR products generated using Taq DNA Polymerase have dA ends. If cloning is the next step, then T/A-cloning is preferred.
- Thaw 2x master mix, Extraction Buffer, primer solutions and mix thoroughly and spin down before use.
- Punch leaf tissue (0.5 to 1.2 mm) and add 8µl FastAmp™ plant direct Extraction Buffer and mix thoroughly
- Prepare a reaction mix according to the following table:
- Mix the reaction mixture thoroughly.
- Program the thermal cycler according to the manufacturer’s instructions. A typical PCR cycling program is outlined in the following table.
- Place the PCR tubes in the thermal cycler and start the cycling program.
- Run 10.0 µl of PCR products in 1% agarose gel (140 volts for 45 min).