Superscript one step rt pcr system for long templates

Total RNA was isolated from 80-ml cultures of Synechocystis strains possessing the xylAB genes at OD₇₃₀ ∼0.7, as previously described (Mohamed and Jansson, 1989 (link)). A Turbo DNA-Free Kit (Thermo Fisher Scientific) was used to remove contaminating genomic DNA. The reverse transcription reactions were carried out using Superscript III enzyme (Thermo Fisher Scientific) and random primers (New England BioLabs). The cDNA molecules thus synthesized were then used as templates for PCR, employing the same set of primers used to amplify the heterologous transporter-catabolic genes. The same primer sets were employed to check the negative controls, in which DNase-treated RNA molecules were used as templates. petA (sll1317), which was used as a positive control, was amplified using the following primers:
5′-ACTCGAATTCATGAGAAACCCTGATACTTTGGGGCTGTGGACGAAAAC-3′ (forward primer) and 5′-ACTCCTGCAGCTAGAAATTAAGTTCGGCAGCTTGAACTTTTTCAATCTG-3′ (reverse primer). For a longer template, i.e., the xylAB genes, the SuperScript One-Step RT-PCR system for long templates (Thermo Fisher Scientific) was used as per manufacturer’s instructions. PCR products were analyzed on a 0.8% agarose gel.

Total RNA was isolated from cultures of Synechocystis strains at OD₇₃₀ ~ 0.7 as previously described (Mohamed and Jansson 1989 (link)). Turbo DNA-free Kit (Thermo Fisher Scientific) was used to remove carried over genomic DNA per manufacturer’s instructions. The reverse transcription reactions were carried out using Superscript III enzyme (Life Technologies) and random hexamers (New England BioLabs). The cDNA molecules were then used as templates for PCR, employing the same set of primers used to amplify the heterologous genes. As negative controls, DNase-treated RNA molecules were used as templates, and the same primer pairs were employed. petA (sll1317) was used as the positive control as described (Ranade et al. 2015 (link)). For longer templates such as araFGH and araBAD gene sets, the SuperScript One-Step RT-PCR system for long templates (Thermo Fisher Scientific) was used per manufacturer’s instructions. PCR products were analyzed on 0.8% agarose gel.