First-Strand cDNA Synthesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Heat the RNA solution to 65ºC for 10 minutes, then chill on ice. Add first-strand cDNA reaction mix and incubate at 37°C for 1 hour

Protocol tips
Heat the RNA solution to 65ºC for 10 minutes, then chill on ice. Add first-strand cDNA reaction mix and incubate at 37°C for 1 hour

Publication protocol

Strains BY4741, Δrck2 and Δrck2 containing plasmids were grown to the mid-logarithmic stage of growth in 4% SD-Trp media 30°C and stressed by the addition of 20% sorbitol for 15 min, rotated at 150 rpm. Cells were broken with glass beads using a MagNalyser (Roche, Burges Hill, UK) bead-beater for 30 s at 4°C, before incubating on ice for 15 min to precipitate proteins. Cell debris and proteins were harvested by centrifugation for 15 min (17,000×g at 4°C). The cell-free supernatant was used for the extraction of total RNA using an isolation kit from Qiagen (Hilden, Germany) and cDNA prepared using a first strand cDNA synthesis kit (GE Healthcare, Bucks, UK). Transcriptional levels were determined by qPCR using the following conditions follows: 0.5 ng/µl cDNA, 6.25 µM forward primer, 6.25 µM reverse primer, 5 µL of 2× SYBR Green master mix (Applied Bio Systems) and made up to 20 µL using molecular grade water

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