Tips & Techniques

ampliPHOX Colorimetric Detection:

For best results, always run a Full System Optimization with each new lot of ampliPHY that you receive and run a System/Reagent Check when opening a new aliquot of ampliPHY if that lot has already been optimized. For more information about the Optimization & Reagent Check, refer to the most recent version of the ampliPHOX Operation Manual.
Be sure to regularly clean out your wash bins and humidity chamber.
The ampliPHY reagents are oxygen sensitive so be sure to keep the vials closed when not in use to avoid excessive oxygen exposure. If you see evidence of polymerization in the vial, do not use the reagent.
If there is evidence of salt precipitation in the ampliTAG Buffer, gently heat the buffer in a 35-39 C waterbath for 5-10 minutes until the salts are dissolved.
Avoid touching the spotted arrays with your hands or pipette tips as this could dislodge spots.
Make sure there are no air bubbles in the solutions applied to the arrays. We’ve found that gently tapping the slide helps to get rid of them.
Always be sure to use the reagents within their expiration.

Custom Arrays:

When designing your arrays, we recommend including multiple replicates of each capture on your array to yield the best results statistically. For our research at InDevR, we typically spot 3 replicates of each capture.
For custom arrays that will be used with ampliPHOX, it’s necessary to include at least 3 control spots to serve as both a positive control for the reagents and to properly align a mask with the ampliVIEW software when analyzing the data.
When you receive your custom arrays, keep them in a dry, dark location at room temperature. Storing the arrays in an area where they will be exposed to moisture and light may compromise your capture probes and ultimately give you unreliable results.

Pre-ampliPHOX Protocol Recommendations:

For nucleic acid microarrays, the target must be biotinylated in order for ampliPHOX to detect. We’ve found the best ways to incorporate biotin is by using biotinylated NTP/dNTP or biotinylated primer(s) in a RT-PCR protocol.
Ideally, a single stranded target of approximately 100-300 bp should be used for your nucleic acid arrays. Therefore, further processing of your target may be necessary for optimal performance. For example, if you are using PCR product that is 1000 bp in length, enzymatically digest the product to create a ssDNA and heat treat to fragment.
For protein microarrays, a block is frequently necessary to avoid non-specific binding. In-lieu of blocking each array individually, incorporate your optimized block buffer in the sample dilution and/or detection antibody dilution.
A water wash is discouraged during the pre-ampliPHOX steps for protein microarrays.
Each assay is unique and optimization of the hybridization, binding, and wash steps as well as the buffers used is necessary to attain your best sensitivity and specificity. We provide some starting points for your pre-ampliPHOX protocol here, but it’s up to you to fully optimize your microarray protocol.

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