Random priming is widely used in first strand cDNA synthesis.
Random Primer Mix is a ready-to-use optimized mixture of hexamers and anchored-dT primer (dT23VN).
A mixture of hexamers and anchored-dT primer provides even and consistent coverage of the RNA template population across a wide range of RNA template concentration. In contrast, traditional random priming by hexamer has poor coverage of 3´ end of RNA templates (1,2,3).
We recommend using Random Primer Mix for reverse transcription of the following RNA templates:
RNA without poly(A) tail such as ribosomal RNAs.
RNA with strong secondary structures.
Partially degraded RNA samples.
Target regions at 5´ end of a long messenger RNA transcript.
Figure 1: Various amounts of human spleen total RNA (2 μg to 20 pg) were reverse transcribed using ProtoScript First Strand cDNA Synthesis Master Mix Kit (NEB #E6300 ) in the presence of 6 μM Random Primer Mix (panel A) or 3.5 μM random hexamers (panel B). About 1/10th of the cDNA products were then analyzed in duplicate qPCR reactions using a pair of primers specific for the beta actin gene with DyNAmo™ FLASH SYBR® Green qPCR Master Mix on a Bio-Rad IQ5® system.
Recommended Usage: Random Primer Mix (60 µM) contains 35 µM hexamers and 25 µM dT23VN and 1 mM dNTPs. We recommend using Random Primer Mix at a final concentration of 6 µM.
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The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the Technical Support Form for appropriate document.)
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