Figure 1.

Generation of the single-strand probe for RPII. T3 and T7 RNA Polymerase promoters flank the multiple cloning site (MCS) of the pBS SK⁺ vector. The direction of transcription of each RNA polymerase is shown in the dotted arrows. Digestion of pBS SK⁺ with BamHI and PstI linearizes the vector, producing sites for cloning of the 1.5-kb A. thaliana RPII insert. The arrow in the insert indicates the direction of transcription of the gene. The 1.5-kb A. thaliana RPII insert lies between the T3 and T7 RNA Polymerase promoters of pBS SK⁺. Digestion of the vector with EcoRI linearizes the vector such that the T3 RNA Polymerase makes a transcript complementary to the transcribed strand of the RPII gene. Digestion of the vector with Xba I linearizes the vector so that the T7 RNA Polymerase makes a transcript complimentary to the non-transcribed strand of the RPII gene. Addition of RNA synthesis reaction components (appropriate RNA Polymerase and radiolabeled NTPs) produces the probes for detection of each of the DNA strands of the RPII insert.