- The template can be amplified by PCR using a primer containing the T7 promoter sequence. In vitro transcription of the PCR product will produce single-stranded, or double-stranded RNA directly if both PCR primers contain the T7 promoter sequence.
- When designing the T7 promoter sequence containing primers, it is recommended to add two Gs after the T7 promoter sequence. Transcription yields have been shown to be reduced if these two Gs are absent.
An example of an amplification primer with T7 promoter (underlined) at the 5´ end preceding the gene-specific (lower-case) sequence is shown below. The first nucleotide of the trancript is indicated (+1).
5´ TAATACGACTCACTATAGGGaaggacagatggttaagtac 3´
The PCR reaction should be analyzed by gel electrophoresis in order to confirm amplification and to provide an estimate of quantification.
- The PCR product can be used directly as a template for transcription, without purification. Alternatively, purify the PCR product by phenol/chloroform extraction and ethanol precipitation, or a spin column (we recommend Monarch PCR & DNA Cleanup kit, NEB# T1030) and resuspend in TE [10 mM Tris-HCl (pH 8.0), 1 mM EDTA, prepared with Milli-Q water or equivalent] to a final concentration of ~500 µg/ml.