NEB - How can I change the initiating sequence of my dsDNA template?

If your sequence of interest is cloned into a plasmid we recommend using the Q5 Site-Directed Mutagenesis Kit (NEB #E0554S) to change the initiating +1 and +2 bases to AG. Before transcription, the plasmid will need to be linearized (using a restriction enzyme that leaves a blunt or 5′ end overhang) to create a double-stranded in vitro transcription template. See the product manual for DNA template preparation.

Alternatively, PCR can be performed using a Forward Primer that is designed to include the T7 promoter (with a few bases upstream) and the G to A change in the initiating nucleotide as well as some downstream sequence. Design the Reverse Primer to anneal to the region where your sequence will be terminated.

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