How do I determine the double-stranded (ds) cDNA yield
How do I determine the double-stranded (ds) cDNA yield
Estimate the yield of ds cDNA using an Agilent 2100 Bioanalyzer:
Open the ds cDNA electropherogram using Agilent 2100 Expert Software.
Choose the ""Region Table"" tab.
Select the expected size range of ds cDNA appropriate for your SMARTer kit. The ds cDNA concentration will be displayed below the graph.
For SMARTer Ultra low kits: select the region encompassing 400–9,000 bp.
For the SMARTer stranded kit: select the region encompassing 150–1,000 bp.
For the SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian: select the region encompassing 200–1,000 bp.
For the SMARTer universal kit: select the region encompassing 100–1,000 bp.
To estimate the total amount of ds cDNA, multiply the ds cDNA concentration (pg/µl or pmol/l) by the volume (µl) of the ds cDNA sample (taking any dilution factor into account).
Evaluating ds cDNA concentration using Agilent 2100 Expert Software. The ""Region Table"" tab is indicated by the green arrow, the selected region is indicated by the blue vertical bars. The cDNA concentration is indicated by the red arrow. cDNA was generated using either 100 pg of Mouse Brain Total RNA spiked with ERCC as input for the SMARTer Ultra Low Input RNA Kit for Sequencing - v3 (Panel A), the SMARTer Stranded RNA-Seq Kit with 1 ng of poly(A)-enriched Human Brain Total RNA (Panel B), the SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian with 1 µg of Control Mouse Liver Total RNA (Panel C), or the SMARTer Universal Low Input RNA Kit for Sequencing with 2 ng of chemically fragmented Human Brain Total RNA (Panel D).