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= EQUIPMENT =
* Incubator (37°C)
* Refrigerator (4°C)
* Freezer (20°C)
* Freezer (70°C)
* Autoclave
* Thermocycler
* Water baths
* Microcentrifuge (preferably three, one for each color-coded area; at
least two are necessary, one for the Grey Area and one for the White
Area)
* Adjustable pipettors (at least three sets of 1000 µL, 200 µL, and 20
µL, one set for each color-coded area; if possible a 0.5–10-µL
pipettor for manipulating enzymes in the White Area)
* Vor tex mixers
* Magnetic stirrer
* Rocker or rotator
* Hot plates
* pH meter
* Vacuum pump (optional)
* Microwave (preferable for melting agarose solutions; however, a hot
plate or Bunsen burner can substitute for this function)
* Power supplies
* UV transilluminator
* Polaroid camera with orange filter
* Hood or camera stand and darkroom
* Bunsen burner
* Analytical balance
= MATERIALS =
* Erlenmeyer flasks (100 mL, 250 mL, 1000 mL)
* Graduated cylinders (100 mL, 250 mL, 500 mL, 1000 mL)
* Beakers (250 mL, 500 mL, 1000 mL)
* Bottles (100 mL, 500 mL)
* Glass pipets (25 mL, 10 mL, 5 mL, 2 mL) and rubber bulbs or Pipette Pumps®
* Sterile Pasteur pipets and rubber bulbs
* Plastic racks for microcentrifuge tubes
* Floating racks for microcentrifuge tubes
* Thermometers
* Squir t bottles for ddH2O, 95% ethanol, 1.5% sodium hypochlorite (bleach)
* 15-mL screw-cap polypropylene tubes tubes and racks
* 50-mL screw-cap polypropylene tubes and racks
* Sterile yellow polypropylene pipet tips (1–200 µL)
* Sterile blue polypropylene pipet tips (200–1000 µL)
* Sterile 1.5-mL microcentrifuge tubes
* Sterile 0.5-mL microcentrifuge tubes
* Sterile 0.2-mL PCR reaction tubes
* 1.5-mL screw-cap microcentrifuge tubes with O-rings
* Parafilm®
* Gloves (disposable latex or vinyl)
* Colored laboratory tape
* Paper towels or Kimwipes®
* Magnetic stir-bars
* Weigh boats
* Spatulas
* Scissors or razor blade
* Permanent markers
* Ice buckets
* Sterile lancets
* Vacuum-sealed blood collection tubes, e.g., Vacutainer® tubes
* Collection vials
* Cotton
* Talc powder (for re-using gloves)
* Sterile toothpicks
* Inoculating loop
* Petri plates
* Gel trays
* Gel boxes
* Combs
* UV protective eyewear (goggles or face shield)
* Polaroid 667 film
* Plexiglass shield (high-grade UV-transmitting Plexiglass)
= GENERAL REAGENTS =
* Boric acid
* EDTA
* Tris base
* Sodium chloride
* Sodium hydroxide
* Sodium acetate
* Potassium chloride
* Potassium acetate
* Bleach (diluted to a final concentration of 1.5% sodium hypochlorite)
* Hydrochloric acid
* Glacial acetic acid
* Triton® X-100
* Glycogen
* Glycerol
* Gelatin
* Sodium sulfate
* Sodium iodide
* Silica particles
* Nitric acid
* Chelex® 100 (Bio-Rad Laboratories, Richmond, CA, No. 143-2832)
* Proteinase K
* Potassium phosphate
* Disodium phosphate
* Sodium dodecyl sulfate (SDS)
* Guanidine isothiocyanate
* Sodium citrate
*β-mercaptoethanol
* Sarkosyl
* Saponin
* tRNA (yeast or E. coli)
* Phenol (equilibrated with water for RNA and equilibrated with Tris
pH 8.0 for DNA)
* Isoamyl alcohol
* Chloroform
* Isopropanol
* 100% ethanol
* Peptone
* Tryptone
* Yeast extract
* Agar
* Agarose
* Ethidium bromide
* Bromophenol blue
* Xylene cyanol
* DNA markers (“PCR markers”):
= SPECIALTY REAGENTS =
* Tryptic Soy Agar (V. cholerae)
* Barium chloride dihydrate for MacFarland’s Standard (V. cholerae)
* Sulfuric acid for MacFarland’s Standard (V. cholerae)
* Saponin (P. falciparum/P. vivax)
* N-acetyl cysteine (M. tuberculosis)
* Sterile phosphate buffered saline (PBS)
* Sodium thioglycolate for Cary-Blair transport medium (E. coli)
* Calcium chloride for Cary-Blair transport medium (E. coli)
* MacConkey agar (E. coli)
* Acetone (Leptospira)
* Sterile Dacron®-tipped applicators (Fisherbrand #14-959-90) (C.
trachomatis/N. gonorrhoeae)
= PCR REAGENTS =
* Primers (Operon Technologies, Alameda, CA; New England Biolabs,
Inc., Beverly, MA; Gibco BRL, Grand Island, NY; Applied Biosystems
Perkin-Elmer, Foster City, CA)
* Nucleotides (dNTPs: dATP, dCTP, dGTP, dTTP)
* 10X Buffers
* MgCl2
* Taq polymerase (see section 5.1.2)
* Reverse transcriptase RAV-2 (Amersham Corp., Arlington Heights, IL)
* rTth reverse transcriptase-polymerase (Perkin-Elmer Corp.)
* Dithiothreitol (DTT)
* Dimethyl sulfoxide (DMSO)
* Tetramethylammonium chloride (TMAC)
* Betaine (Sigma Chemical Co., St. Louis, MO, B-2629)
* Bovine serum albumin (BSA)
* Manganese acetate
* Bicine
* Nuclease-free sterile ddH20
* Mineral oil
* Wax beads
= ADDITIONAL MATERIALS AND REAGENTS FOR NONRADIOACTIVE PROBES =
* Dot blot microfiltration apparatus
* Rocker/orbital shaker
* “Seal-a-meal”® for sealing hybridization bags
* Plastic bags for hybridization
* Plastic dishes with covers
* Filter forceps
* Digoxigenin-11-dUTP, alkali-labile (Boehringer Mannheim,
Indianapolis, IN, No. 1573152)
* Anti-digoxigenin alkaline phosphatase conjugate (Boehringer
Mannheim, No. 1093274)
* Nonfat milk powder
* Blocking Reagent (e.g., Boehringer Mannheim, No. 1096176)
* Nylon membranes (e.g., Boehringer Mannheim)
* Whatman 3MM paper
* Nitrotetrazolium blue (NBT)
* 5-bromo-4-cloro-3-indolyl-phosphate, toluidine salt (BCIP)
* For chemiluminescent detection:
* Darkroom setup (trays, wooden forceps, etc.) or automatic developer
for X-ray film
* Lumi-phos 530® (chemiluminescent substrate; Lumigen, Inc., Southfield, MI)
* Plastic sheets
* X-ray film
* film cassettes
* GBX developer and fixer
= ADDITIONAL MATERIALS AND REAGENTS FOR CLONING =
* Metal block for microcentrifuge tubes (precooled to 20°C)
* Centrifuge with 3500-rpm capability
* Spectrophotometer measuring optical density at 600-nm wavelength
* Plate spinner, if available
* Air shaker (37°C)
* Plate spreader (i.e., made from a glass rod)
* Sterile wooden sticks
* Ampicillin
* Potassium acetate
* Glucose
* Polyethylene glycol (MW 8000)
* Polyethylene glycol (MW 3350)
* Magnesium sulfate
* 5-Bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal)
* Isopropyl β-D-thiogalactopyranoside (IPTG)
* Dimethyl formamide (DMF)
* Cloning vector (e.g., Bluescript, Stratagene Cloning Systems)
* Restiction enzymes
* Restriction enzyme buffers
* T4 DNA Ligase
* 10X T4 DNA Ligase Buffer
* RNase
* Transparent tape
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