SMARTer® PCR cDNA Synthesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Produces high-quality cDNA from nanograms of total or poly A+ RNA. Store RNA and SMART II A Oligo at –70°C. Store all other reagents at –20°C	For ds cDNA Polishing Proteinase K treatment is necessary to inactivate the DNA polymerase activity before proceeding with the ligation steps. Do not chill the tube at –20°C or on ice before centrifuging. Chilling the sample will result in coprecipitation of impurities

Upstream tips
Produces high-quality cDNA from nanograms of total or poly A+ RNA. Store RNA and SMART II A Oligo at –70°C. Store all other reagents at –20°C
Protocol tips
For ds cDNA Polishing Proteinase K treatment is necessary to inactivate the DNA polymerase activity before proceeding with the ligation steps. Do not chill the tube at –20°C or on ice before centrifuging. Chilling the sample will result in coprecipitation of impurities

Publication protocol

Total RNA was extracted using the following conditions: (1) P. lutzii yeast cells maintained in MVM without Cu; and (2) yeast cells cultivated in MVM without Cu, incubated for 3 h at 37°C followed by the addition of the different ECM components (laminin, fibronectin, and types I and IV collagen) at 50 μg/ml, and incubated for an additional 2 h at 37°C. These preps were used for the Real Time PCR assays.

Total RNA was extracted from all experimental conditions using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA). First-strand cDNA synthesis was performed using reverse transcriptase (RevertAid™ H Minus Reverse Transcriptase, Fermentas Life Sciences, Canada) and 1 μg of total RNA. First-strand cDNA was used as a template to synthesize the second-strand of cDNA using the SMART PCR cDNA synthesis kit (Clontech Laboratories, Palo Alto, CA, USA).

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Manufacturer protocol

Download the product protocol from Takara Bio Inc for SMARTer® PCR cDNA Synthesis Kit below.

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