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.

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.