What are abnormal Bioanalyzer traces for all SMARTer kits?

1. Elevated baseline in the Bioanalyzer trace. This is commonly due to the presence of SPRI beads in the cDNA preparation. Although SPRI beads themselves do not fluoresce (nor will they bind the dye included in the Agilent High Sensitivity DNA Kit), any DNA remaining on the bead will bind dye and fluoresce. Electropherograms of magnetic bead-contaminated cDNA sample (Panel A) and the same [...]

Can I substitute alternative products for any of the recommended additional materials?

SMARTer kits are based on complex technology and require precise adherence to the experimental procedure. Each step of the protocol, including equipment, has been carefully optimized. Nuclease-free thin-wall PCR tubes (0.2 ml; USA Scientific Cat. # 1402-4700) have the lowest affinity for RNA, DNA, and SPRI beads. Using strip tubes ensures better reproducibility between multiple samples and [...]

How can I ensure efficient cDNA purification using SPRI beads?

To ensure that purification of cDNA using SPRI beads occurs efficiently throughout the protocol, use the magnetic device specifically recommended for each type of tube. If the protocol requires multiple purifications, do not use the same magnetic device for all steps. Aliquot SPRI beads prior to use to avoid cross-contamination. Bring SPRI-bead aliquots to room temperature prior to purification [...]

Why do I have to use different magnetic devices for SPRI bead purification of cDNA?

The following magnetic devices, recommended in the user manuals for some SMARTer cDNA synthesis kits, have been validated for SPRI bead-based purification of cDNA in different types of tubes. You may also make your own magnetic separator using rare earth magnets. For nuclease-free thin-wall PCR tubes (0.2 ml; USA Scientific, Cat. # 1402-4700): SMARTer-Seq Magnetic Separator - PCR Strip, Cat. [...]

Why do I have to perform a negative control during SMARTer cDNA synthesis?

A negative control (performing the entire cDNA synthesis and purification procedure in the absence of any RNA input, but maintaining the same reaction volume) is essential for the evaluation of cDNA synthesis as well as for identifying potential problems, including contamination. We recommend performing a negative control reaction each time the protocol is performed, especially when using the [...]