DeCodiFi™ Plant Direct

Yes. DeCodiFi™ Plant Direct is designed to enable direct amplification from crude plant extracts, eliminating the need for DNA purification. A crude extract preparation step is still required before PCR.

You may use your standard lysis protocol or the recommended HotSHOT lysis protocol described in the DeCodiFi™ Plant Direct Technical Data Sheet. This protocol uses 1–4 mm² of plant tissue incubated in an alkaline lysis solution at 95°C, followed by the addition of a neutralization solution. The resulting lysate can be used directly for PCR. Both the lysis and neutralization solutions may be prepared by the user or provided upon request with the purchase.

For a 25 µL reaction, use 1.25–5 µL of plant crude extract, corresponding to 5–20% of the total reaction volume. Most plant species perform well using 20% HotSHOT extract in the PCR reaction.

For plants with high inhibitor levels, such as strawberry leaves, the protocol recommends diluting the HotSHOT extract 1:4 with nuclease-free water and continuing to use 20% of the diluted extract in the PCR reaction.

The mix includes DeCodiFi™ Hot-Start High-Fidelity Polymerase, dNTPs, magnesium, enhancers, and proprietary stabilizers. Primers and template DNA are not included.

Yes. Because DeCodiFi™ has strong 3′-exonuclease proofreading activity, the protocol recommends incorporating two phosphorothioate bonds at the 3′ ends of primers. This helps prevent primer degradation, enhances specificity, and reduces primer dimer formation. Use primers of 20–28 nt in length, 40–60% GC content (preferably 50–60%), and similar Tm values (≥60°C). Avoid 3′ complementarity, long G/C runs, and secondary structures.

The recommended PCR program starts with 95°C for 2 minutes, followed by 30–40 cycles of 95°C denaturation for 10 seconds, annealing at the calculated Tm for 15 seconds, and extension at 72°C for 15 sec/kb. For GC-rich templates above 70% GC, the protocol recommends using 98°C for 10 seconds during denaturation.

Yes. A gradient PCR is strongly recommended when setting up cycling conditions. Use the lower primer Tm as a reference, starting 6°C below that value and increasing in 2°C increments up to 6°C above the calculated Tm.

Yes. DeCodiFi™ Plant Direct is compatible with multiplex PCR. The protocol recommends validating each primer pair individually before multiplexing, using primers with similar Tm values, starting with 0.05 µM of each primer, and adjusting primer concentration, annealing temperature, cycle number, and extension time based on the longest fragment in the mix.

DeCodiFi™ Plant Direct is recommended for direct PCR, genotyping by sequencing, and transgene detection from plant material.

Store DeCodiFi™ Plant Direct at -20°C upon arrival and avoid repeated freeze-thaw cycles. The mix is designed to be transported at 2–8°C without loss of performance for up to 7 days.