Protoscript rt pcr kit

For the expression analyses illustrated in Figures 4A, 7B and 8A, cells were cultivated to an A₆₀₀ of ∼0.5 in a 125 ml culture at 30°C and 15 ml aliquots were removed and subjected to an instantaneous 30 to 39°C upshift for the indicated times. Heat shock induction was terminated through addition of 20 mM sodium azide, and RNA was isolated as above. For induction of YFR057w (Figures 9A, S4), cycloheximide was added to a final concentration of 0.2 mg/ml, 15 ml aliquots were removed at the indicated times and RNA was isolated.
Contaminating genomic DNA was removed from each RNA sample by digestion with RNase-free DNase I (OMEGA Bio-tek Inc #E1091), followed by phenol/chloroform extraction. 0.5 µg purified RNA was used in each cDNA synthesis with ProtoScript RT-PCR Kit (NEB #E6400S). Oligo(dT) primers were used in cDNA synthesis for quantification of Pol II gene transcripts; random primers were used in cDNA synthesis for quantification of SCR1 RNA. 2% of the synthesized cDNA was used in each qPCR, which was performed as described above. Primers were designed to target the 3′UTR of HSP82, YFR057w and PMA1, or the body of SCR1. Their coordinates are: HSP82, +2134 to +2228; YFR057w, +312 to +437; PMA1, +2998 to +3083 and SCR1, +385 to +483. For quantification, SCR1 was used to normalize HSP82 and YFR057w mRNA levels.

The DNA was extracted for previous phylogenetic studies from fresh or dried leaf material, using a CTAB-based method [48 ]. RNA was extracted from fresh leaf material harvested from reproductively mature plants (Table 1). Plant tissue was snap-frozen in liquid nitrogen and total RNA was extracted using a commercial extraction kit (Promega), following the manufacturer’s instructions. Crude total RNA preparations were treated with TURBO DNA-free™ (Ambion) to remove residual DNA. RNA quality was inferred by running 5 μl on an agarose gel, and RNA concentrations were estimated using a NanoDrop Spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). Prior to cDNA synthesis, the presence/absence of genomic DNA contamination was tested for all RNA preparations using PCR reactions with PIF and Pong primers known to work on genomic DNA, and an RT- control supplied with the cDNA synthesis kit. cDNA was generated from DNA-free total RNA using the Protoscript RT-PCR kit (NEB) using 2 μl of random and oligo-dT primers and following the manufacturer’s protocol. The cDNAs were used as templates in amplification reactions as described below.