- The T3 promoter primer is a short, single-stranded oligonucleotide designed to anneal to the T3 promoter sequence, which serves as the binding site for T3 RNA polymerase.
- The T3 promoter is widely used in cloning vectors due to several key advantages that make it an efficient tool for RNA synthesis, sequencing, and gene expression studies.
- The T3 promoter primer sequence corresponds to the conserved T3 RNA polymerase recognition site, ensuring efficient transcription initiation. The T3 promoter primer is typically 20 nucleotides long, although some variations may include additional bases downstream to optimize transcription efficiency.
- The canonical sequence for the T3 promoter primer is:
- 5′-AATTAACCCTCACTAAAGGG-3′
- The melting temperature (Tm) of the standard 20-base T3 promoter primer is approximately 50–55°C, depending on reaction conditions and buffer composition. The GC content is around 40% (with 8 G/C bases and 12 A/T bases). The annealing temperature typically ranges from 48–55°C, which can be adjusted depending on the specific needs of the PCR setup. For high-specificity applications such as in vitro transcription (IVT), mRNA synthesis, or sequencing, the annealing temperature can be optimized to 50–52°C.
- The T3 promoter primer is used in a variety of applications, particularly for transcription, amplification, and sequencing where the T3 promoter is involved.
- In general, desalting purification is suitable for most applications, but for critical procedures like RNA synthesis, HPLC, or PAGE purification may be recommended to maximize the purity and efficiency of the primer.
