Home FAQs How can I change the initiating sequence of my dsDNA template?

FAQ: 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.