Chromo4 real time pcr machine

Reporter gene analysis was performed in the F3 generation after genetic crossing. To detect β-glucuronidase (GUS) activity, 5-day-old seedlings were incubated in reaction buffer containing 0.1M sodium phosphate buffer (pH 7), 1mM ferricyanide, 1mM ferrocyanide, 0.1% Triton X-100 and 1mgml⁻¹ X-Gluc for 10 min to 8h in dark at 37°C. Afterwards, chlorophyll was removed by destaining in 70% ethanol and seedlings were cleared with 70% (w/v) chloral hydrate–10% (v/v) glycerol solution. Analysis of fluorescence reporter expression was performed using a Zeiss LSM780 confocal microscope. Counterstaining of root cell walls was achieved by mounting roots in 10 µM propidium iodide (PI).
The RNeasy Plant Mini Kit (Qiagen) was used for RNA extraction from roots. RNA was treated with DNase (Fermentas) and transcribed into cDNA using SuperScriptII (Invitrogen). Quantitative reverse transcription PCR (qRT-PCR) was performed in triplicates using the Mesa Blue Sybr Mix (Eurogentec) and a Chromo4 real-time PCR machine (Bio-Rad). Oligonucleotide sequences are given in Supplementary Table S1 at JXB online. Expression levels were normalized to the reference gene At4g34270 (Czechowski et al. 2005 (link)). The JLO misexpression experiments were performed as described in (Rast and Simon, 2012 (link)).

Primers were designed using the tool available at Invitrogen (http://tools.lifetechnologies.com/content.cfm?pageid=9716) to give a product between 70 and 200 bp. Primer sequences are the following: lsrB forward (F): (5′-CCCAGTGTTTCTGGTCAGGT-3′) and reverse (R): (5′-AACCGCAGAAACGATAATGG-3′), lsrK F:(5′-TCGACACCTATACGCTGCTG-3′) and R:(5′-CGCAGGTGATACCAGGTTTT-3′), dinB F:(5′-ACGCCTACAAAGAAGCCTCA-3′) and R:(5′-TTGCAGCTCGTTGAAGATTG-3′), umuC F:(5′-TGGGGGATTTCTTCAGTCAG-3′) and R:(5′-TTCCTCTGCCCTCTTTAGCA-3′). The duration of the reverse transcription reaction was 60 min at 45 °C, the reaction was stopped at 95 °C for 15 min. Reverse transcription products were subjected to 50 cycles of PCR amplification (1 min at 95 °C for denaturation, 1 min at 62 °C for annealing and 30 s at 72 °C for extension). At the end, we run a dissociation curve by gradually increasing temperature from 55 to 95 °C (0.2 °C per second). The iScript One-Step RT-PCR Kit with SYBR Green was used. All reactions were performed with Bio-Rad (M J Research) Chromo4 real-time PCR machine, and we used Opticon Monitor 3 for analysis.