Transcriptor First Strand cDNA Synthesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add 0.5 ul (10 U) of Reverse Transcriptase to the reaction mix and incubate for Incubate 30 min at 55°C. Inactivate Transcriptor Reverse Transcriptase by heating to 85°C for 5min

Protocol tips
Add 0.5 ul (10 U) of Reverse Transcriptase to the reaction mix and incubate for Incubate 30 min at 55°C. Inactivate Transcriptor Reverse Transcriptase by heating to 85°C for 5min

Publication protocol

DNA and RNA experiments.
Chromosomal DNA from P. brassicacearum NFM421 was prepared by phenol-chloroform extraction. Plasmids were extracted with the QIAprep spin miniprep kit (Qiagen), and DNA fragments were purified from agarose gels with the QIAquick gel extraction kit (Qiagen) according to the manufacturer's instructions. RNA was extracted using the RNAprotect bacteria reagent and RNeasy mini kit (Qiagen). Reverse transcription-PCR (RT-PCR) assays were done using the Transcriptor first strand cDNA synthesis kit (Roche).

5′ RACE.
The 5′ ends of rsmX and rsmY transcripts were mapped by rapid amplification of cDNA ends (RACE). A reverse transcription reaction was performed using total RNA from P. brassicacearum strain NFM421 isolated in late stationary phase and the Transcriptor first strand cDNA synthesis kit (Roche). The 5′-phosphorylated, 3′-end cordecypin-blocked oligonucleotide DT88 (37) was ligated to the single-stranded cDNA with T4 RNA ligase (New England BioLabs). The anchor-ligated cDNA was amplified first with primers DT89 (anchor-specific primer) and an rsm-specific primer (see Table S1 in the supplemental material). Then, a nested PCR was carried out using DT89 and a second rsm-specific internal primer. Finally, the PCR product was cloned into the cloning vector pCR4Blunt-TOPO (Invitrogen). Three independent clones were sequenced using primer T7 (GATC Biotech).

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