Equipment and Materials

Growing and harvesting Arabidopsis leaves

• Arabidopsis seeds (Arabidopsis thaliana, accession Col-O)
• 10 cm square pots
• Miracle-Gro Potting Mix (Scotts Company LLC, Bergen, NY)
• Window screen mesh (New York Wire, Mt. Wolf, PA)
• Conviron E15 growth chamber (Conviron, Winnipeg, Canada) set at 23°C, 60% relative humidity, and 120 µmol/m²sec light with a long-day photoperiod (14 hr light, 10 hr dark)
• Cardboard and black 55-gallon trash bags
• 1.5 ml microcentrifuge tubes
• Liquid N₂

Week 1

Total RNA isolation and quantification

• Qiagen RNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA)
• RNase-free water
• NanoDrop™ One microvolume UV-Vis spectrophotometer (Thermo Scientific, Wilmington, DE) or similar

Formaldehyde gel electrophoresis

All solutions and glasswares are treated with 0.1% DEPC (Diethyl pyrocarbonate) as described in Appendix B.

• RNA Millenium™ Markers (Thermo Fischer Scientific, Waltham, MA)
• Agarose (Thermo Scientific, Wilmington, DE )
• 20X F.A. gel buffer (400 mM 3-[N-morpholino]propanesulfonic acid or MOPS, a Good’s buffer; 100 mM sodium acetate; 20 mM EDTA, pH to 7.0 with NaOH)
• 1X F.A. gel running buffer (50 ml 20x F.A. gel buffer; 20 ml 37% (12.3 M) formaldehyde and bring up to 1 liter with RNase-free water treated with DEPC (See Appendix A)).
• 5X RNA loading buffer (16 µl saturated aqueous bromophenol blue solution; 80 µl 500 mM EDTA, pH 8.0; 720 µl 37% (12.3 M) formaldehyde; 2 ml 100% glycerol; 3.084 ml formamide; 4 ml 10X F.A. gel buffer and bring up to 10 ml with RNase-free water)
• GelRed (Phenix Research Products, Candler, NC)
• Mini-Sub Cell G.T. Systems and power supply (Bio-Rad, Hercules, CA)

Week 2

Reverse transcription, the first strand of cDNA synthesis

• Verso cDNA Synthesis Kit (Thermo Scientific, Wilmington, DE)
• Individual 0.2 ml thin wall PCR tube (Bio-Rad, Hercules, CA)
• CFX Connect™ Real-time PCR Detection System (Bio-Rad USA, Hercules, CA) or similar
• Ice chests

qPCR

• Strips of eight 0.2 ml thin wall PCR tubes per strip (Bio-Rad, Hercules, CA)
• CFX Connect™ Real-time PCR Detection System (Bio-Rad USA, Hercules, CA) or similar
• GoTaq qPCR Master Mix (Promega US. Madison, WI)
• qPCR primer sets (Table 4-1)