Dicistronic RNA templates were made by digesting the cellular reporter vectors downstream of the firefly luciferase coding sequence and utilizing a T7 promoter incorporated in the vector.
Linear template DNA was generated by digesting the plasmid with restriction enzyme NotI and RNA was produced by in-vitro transcription using T3 RNA polymerase.
The PCR mixture was then cut with the enzyme DpnI to digest the template mSlo1.
The Restriction enzyme DpnI was added to the PCR reaction mixture to digest the template.
The PCRs were treated with the restriction endonuclease DpnI to digest the template DNA, and transformed into E. coli.
The step, however, can easily be done in a high throughput manner by digesting templates in 96- or 384-well format prior to PCR and even the additional amount of DNA required would allow at least 1000 genes to be screened with the present DNA yield (1 μg/individual plant).
Before the preparative digest, conditions were optimized by digesting 1 μg of template DNA with a range of DNase I concentrations (Fermentas, Waltham, MA, United States).
