Can I analyze unpurified double-stranded (ds) cDNA for PCR cycle optimization?
Can I analyze unpurified double-stranded (ds) cDNA for PCR cycle optimization?
PCR-amplified ds cDNA can be analyzed directly from the PCR reaction mix, prior to SPRI bead purification, using an Agilent 2100 Bioanalyzer. The ds cDNA profile will contain a large peak immediately following the Lower Marker; this represents the primer or primer dimers. The Bioanalyzer software may assign the primer/primer-dimer peak as the Lower Marker. If this occurs, manually reassign the Lower Marker.If the ds cDNA yield is low, you may further amplify the cDNA, using several additional PCR cycles, before continuing with purification with SPRI beads as described in the protocol.
Note: If you are using a kit that includes a SPRI bead purification step prior to PCR amplification in the protocol, pipette the cDNA sample carefully to ensure that SPRI beads are not introduced into the Agilent 2100 Bioanalyzer.
Electropherograms of unpurified, PCR-amplified DNA. Panel A shows a negative control, and Panel B shows a positive control generated with 15 cycles of PCR. The green arrow indicates the primer/primer-dimer peak.