Materials and Reagents

I.

• 95% Ethanol (Sigma E7023 or equivalent)
• DNase/RNase free water (Life Technologies AM9937 or equivalent)
• Deionized/Distilled water
• NaCl (Sigma S3014 or equivalent)
• NaOH (Sigma S5881 or equivalent)
• HCl (Sigma H1758 or equivalent)
• Glycine (Sigma G7126 or equivalent)
• Threonine (Sigma T8375 or equivalent)
• Guanidine Isothiocyante (Fisher Scientific 15535-016 or equivalent)
• Chloroform(Sigma C2432 or equivalent)
• 6 N HCl (Sigma XX0628 or equivalent)
• 3 N HCl (Fisher Scientific 18-610-903 or equivalent)
• pH strips (Fisher Scientific 88-841 or equivalent)
• 10X PBS tissue culture (t.c.) grade (Sigma P5493) to be diluted to 1X
• KCl (Sigma P9541 or equivalent)
• Potassium Dihydrogenphosphate (Sigma P9791 or equivalent)
• Disodium Hydrogen Phosphate (Sigma S5011 or equivalent)
• Glycerol (Sigma G5516 or equivalent) Reagent specific for Section A5
• Potassium Citrate (Sigma P1190 or equivalent) Reagent specific for Section A2
• Beef extract powder (Sigma B4888 or equivalent) Reagent specific for Section A2
• Tris Base (Fisher Scientific BP152-1 or equivalent) Reagent specific for Section A2
• Pectinase (Fisher Scientific ICN19897910 from Aspergillus niger or equivalent) Reagent specific for Section A2
• DNase/RNase-free microcentrifuge tubes, non-stick, low retention, siliconized 0.5 ml (Life Technologies AM12350 or equivalent)
• DNase/RNase-free microcentrifuge tubes 1.5 ml, non-stick, low retention, siliconized (Life Technologies AM12450 or equivalent)
• DNase/RNase-free microcentrifuge tubes 2.0 ml, non-stick, low retention, siliconized (Life Technologies AM12475 or equivalent)
• Filter barrier aerosol resistant micropipettor tips DNase/RNase free (0.2 – 1000 µl)
• Qiagen QIAamp Viral RNA Mini Kit (Qiagen 52904)
• Qiagen RNeasy Mini Kit (Qiagen 74104) Reagent specific for Section A3
• Qiagen QIAshredder (Qiagen 79654) Reagent specific for Section A1, A2, A4
• Qiagen collection tubes (Qiagen 19201)
• OneStep™ PCR Inhibitor Removal Kit (Zymo Research D6030)
• 2.0 mL microcentrifuge tubes DNase/RNase free (USA Scientific 1620-2799 or equivalent)
• Primer TE (formula provided Section A6)
• OneStep RT-PCR Kit (Qiagen 210210 or 210212)
• Ambion Superase·In RNase Inhibitor (20 units/µl); Life Technologies AM2694 (2,500 U) or AM2696 (10,000 U)
• 50 mM MgCl2 (BioRad 1708872 or equivalent) or 25 mM MgCl2 (ThermoFisher Scientific AB0359, or equivalent)
• Internal Control RNA (BioGX Cat No. 750-0001—contact company)
• Positive controls (Quantitative Synthetic Norovirus GI— ATCC VR-3234SD, Quantitative Synthetic Norovirus GII—ATCC VR-3235SD, HAV—ATCC VR-1402 and murine norovirus ATCC VR 1937)
• Fluorescein calibration dye (BioRad 1708780 or equivalent)
• Extraction control murine norovirus (ATCC VR 1937) Mengovirus (CECT Spanish Type Culture Collection vMCO CECT 100000 Spanish ATCC)
• Standard desalted primers and HPLC probes for all RTqPCR assays (Integrated DNA Technologies or equivalent)

Equipment and Supplies

II.

• pH meter (Cole Parmer EW 53026-54 or equivalent)
• Biological Safety Cabinet (BSC- 2 Type A2 or higher air exchange rate)
• Ultra low freezer (-70 ˚C or lower)
• Latex or nitrile gloves (powder-free)
• Vortex mixer (Labsource S16-109 or equivalent)
• Nasco Whirl PakTM filter bags;6” x 9” (Fisher Scientific B01348WA or equivalent)
• DNase/RNase-free microcentrifuge tubes, non-stick, low retention, siliconized 0.5 ml (Life Technologies AM12350 or equivalent)
• Sterile scissors
• Ice Bucket and crushed ice
• Hype-Wipe Disinfecting Towelettes (Fisher Scientific 14-412-56 or equivalent)
• Adjustable Micropipettors (0.2 – 1000 µl), dedicated for RNA work only
• Filter barrier aerosol resistant micropipettor tips DNase/RNase free (0.2 – 1000 µl)
• AB 7500 FAST (Applied BioSystems 4351106)
• ABI 96 well plates (Life Technologies 4346906)
• ABI plates cover (Life Technologies 4311971)
• Mini Plate Spinner (Fisher Scientific 14-100-143 or equivalent)
• Mini Centrifuge (Labsource C90-044 or equivalent)
• 96 well cool rack (Sigma Aldrich #Z606634-1EA or equivalent)
• 500 ml Blender (Cole Parmer EW 04243-25 or equivalent) Equipment specific for Section A3
• Blender Base (Fisher 14-509-19, or equivalent) Equipment specific for Section A3
• Disposable scalpels (Fisher Scientific 12-460-456 or equivalent) Supplies specific for Section A3
• 50 ml polypropylene conical tubes (Fisher Scientific 14-959-49A, or equivalent)
• 150 mm x 15 mm Petri Dishes (Fisher 08-757-148 or equivalent)
• 50 mL serological pipettes (Labsource K79-156-CS or equivalent)
• Mettler Toledo™ NewClassic ME Precision Balance, 2200 g (Fisher Scientific 01-912-408 or equivalent)
• Drummond pipette aid (Fisher Scientific or equivalent)
• Refrigerated centrifuge capable of speeds of up to 12,000 x g, and with rotors capable of holding 50 ml conical tubes (ThermoFisher Scientific 75006591or equivalent)
• FY14 50 carbon fiber rotor (ThermoFisher Scientific 46922 or equivalent)
• 70 ml polypropylene ultracentrifuge tubes (Fisher Scientific 01-013-33 or equivalent)
• Ultracentrifuge capable of speeds of 170,000 x g and with rotors capable of holding 70 ml ultracentrifuge tubes (ThermoFisher Scientific 75000100 or equivalent)
• Centrifuge capable of speeds of 21,000 x g, and with rotors capable of holding 1.5 ml and 2.0 ml microcentrifuge tubes (Eppendorf 2231000655 or equivalent)
• Disposable transfer pipettes (Fisher 13-711-22 or equivalent)
• Orbital shaker (Fisher Scientific 11-676-231 or equivalent)