If you are currently isolating DNA from a seedling, nursery palm or field planted palm, your current method should be compatible with the SureSawit™ SHELL Gel Kit. We have tested various home brew and commercially available DNA isolation kits and all have worked well with the kit. As with any PCR based reaction, the higher the purity of your DNA the better the reaction results. Our recommendation is the CTAB method (such as Doyle JJ, Doyle JL: A rapid isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 1987, 19:11-15. or Doyle JJ, Doyle JL: Isolation of plant DNA from fresh tissue. FOCUS 1990, 12:13-15.) or the Qiagen DNeasy Mini Plant kit (Cat No. 69104).

DNA was extracted using the CTAB method, samples A and B, or the Qiagen DNeasy Mini Plant kit (Cat No. 69104), samples C and D. dura (sample D) and tenera (samples A, B and C) fruit form were identified regardless of DNA purification method. Sample E is a no DNA control.

We recommend that you start with 10 ng of input DNA depending on the purity of your sample. A less pure sample could require an additional clean up or a lower concentration of input DNA to reduce endogenous sample PCR inhibitors. We have had success using sub-ng amounts of seedling DNA when rapid, more affordable, extraction methods were used (in development). When DNA amount is too much, occasionally you will observe a lesser intense band of same size from the other allelic sequence.

In the picture above, we purified DNA with the Qiagen DNeasy Kit and amplified from 320 ng to 1.25 ng. Higher concentrations of DNA gave non-specific banding patterns while lower concentrations showed reduced band intensity. 10 and 20 ng of input DNA gave consistent genotype specific band patterns.

Cycling times and temperatures have a direct impact on yield and primer binding for accuracy of results. We have rigorously tested the cycling parameters of the PCR reaction to optimize the time and temperatures with the primer design and master mix formulation. We do not recommend varying the number of cycles or the cycling temperatures.

MP and AP control DNA was amplified for increasing cycle times. We found that 35 cycles produces the most robust bands while reducing primer dimer formation.

Positive controls were amplified with increased annealing temperature. We found that 58.0 °C gave the most robust bands without generating non-specific amplification.

If you see ghost bands or non-specific priming follow these steps:

1. Ensure that the ghost bands are not due to an error during gel loading where a small amount of a positive well leaked into a negative well. If SYBR green detection was used, check the PCR plate picture for increased background in wells corresponding to samples with ghost bands. If background is low than the ghost band could be due to a gel loading error.
2. Check the negative control to ensure that no bands are present. Ghost bands in negative controls and samples could be the result of cross contamination from positive controls, other samples or other primer mix within the sample. See FAQ for bands in negative control.
3. Reduce input DNA. Too much DNA can cause ghost bands or other non-specific priming (smearing or primer dimers). 10 ng is recommended but reducing the DNA input to 5 ng or below is acceptable.
4. Keep reactions on ice during entire assembly process especially if not performed rapidly, or for assembly of multiple plates at a time. At warmer temperatures, but below the primer annealing temperature of 58°C, non-specific amplification due to false priming could occur as the Taq Polymerase does not require heat activation. For this reason starting PCR plate at 94°C directly from ice may also reduce ghost band and primer dimer occurrence.
5. Reduce the number of cycles. We recommend 35 cycles, but reducing the number of cycles to 30 or 31 can help eliminate ghost banding.

1. One likely source of contamination in the negative control wells is the positive control. Be sure to spin down controls before opening. Do not blow-out tips (over-depress pipette) that have been used for pipetting of positive control and discard tips at a safe distance from open PCR plates. It may also be helpful to change gloves or wash hands after handling positive controls.
2. Cross contamination from adjacent sample wells can also cause bands in the negative control wells, especially if the same tip is used for adding the primer mix cocktail across wells.
3. Contamination could come from the environment, which can be reduced by covering PCR plates with a pipette tip box lids during assembly between reagent additions.
4. One environmental source of contamination could be previously run PCR products, so avoid handling open completed PCR plates in the location where PCR assembly is performed.
5. Cross contamination of the DNA source plate could occur during removal of the plate seal. Ensure that the PCR plate is held firmly in place by inserting it into an empty tip box. This will prevent the plate from springing and snapping during seal removal and causing micro-droplet splatter between wells.
6. If bands are present in the negative control reactions, then there is a high probability that the samples are also contaminated. Samples should be rerun until negative controls are void of bands.

1. What DNA purification method are you using? If your DNA is not of sufficient purity you could see faint or missing bands of interest. Try cleaning up your DNA; we recommend the DNA Clean & Concentrator-5 (Zymo Research, Cat no. D4013 ) or use the CTAB protocol or Qiagen DNeasy kit (Cat no. 69104) for initial DNA purification. Alternatively try further diluting DNA in nuclease free water. Crude DNA extractions can contain a greater concentration of PCR inhibitors. Diluting low purity DNA even below 1 ng/uL will intensify faint or missing bands of interest.
2. Letting the PCR reaction sit for an extended period of time in a warm (>23 °C) environment may reduce band of interest, while increasing non-specific amplification (smear and primer dimer amplification). If this occurs, keeping reactions cold during set up will intensify the bands of interest.
3. Some DNA elution buffers can contain high levels of EDTA (DNeasy kit buffer AE has an EDTA concentration of 0.5 mM), which can chelate the Mg+2 ions needed by the Taq Polymerase for PCR. We recommend further diluting eluted DNA in Nuclease-free water (2-5 fold dilutions) or Low EDTA (0.1 mM) TE Buffer (>5 fold dilutions).
4. Numerous freeze thaws on the primers and master mix can decrease the intensity of the band of interest and increase non-specific PCR (smearing and primer dimers).

The components should give good results for up to 8 freeze/thaw cycles. We recommend as few freeze/thaws as possible for optimal performance. If more than 8 freeze thaw cycles are planned than split components into extra aliquots prior to refreezing.

You can follow the SYBR^® Green dye detection protocol for a quicker analysis of your data. We recommend running an agarose gel on any samples with atypical genotypes or questionable intensities by SYBR® Green detection.

Samples were amplified with the SureSawit SHELL Gel Kit and analyzed with either the standard gel protocol or the SYBR^® Green protocol. +/- patterns were identical in both methods allowing for accurate dura, tenera or pisifera indentification.

You can use the kit with your unique palm crosses, but the banding pattern may not follow the same pattern as found with the Deli dura genetic profile. We have tested different palm species and sometimes you will not observe an amplification from each primer mix. Sometimes, you will see a band of different size due to deletion or insertion within the amplified region of the genomic DNA.