Equipment & Reagents Required for Pulsed Field Gel Electrophoresis (PFGE)

Equipment & Reagents Required for Pulsed Field Gel Electrophoresis (PFGE)

Equipment and Supplies Needed for the Molecular Subtyping of Foodborne Bacterial Pathogens by PulseNet’s Standardized PFGE Protocols

(Approximate Cost in US$ as of June 2, 2010)

Electrophoresis and Documentation Equipment:

Other Equipment:

Incubator ($1,000 to $5,000)
Water baths (two are desirable) (~$650.00 for one)
Orbital/shaker water bath or shaker incubator (lysis and washing plugs)(~$3,500 to $7,000)
Optical density instruments (cell suspension adjustment):
Dade Microscan Turbidity Meter (Dade Behring; cat# B1018-66) (~$1,000)
OR
Spectrophotometer (~$1,200 to $3,000)
OR
bioMérieux Vitek Colorimeter with standards (~$600.00)
Scales/balances to measure solid reagents (~$650 to $1,200)
Microwave with turntable (melt agarose)($100 – $200)

Supplies and Selected Reagents:

SeaKem Gold Agarose (Cambrex Research Products)
25 grams (catalog number 50152) (~$138)
125 grams (catalog number 50150) (~$505)
PFGE plug molds (Bio-Rad)
50-well disposable (1.5-mm x 10-mm x 5-mm, Bio-Rad 170-3713) (~$45) OR
Three to five 10-well reusable (2-cm x 1-cm x 1.5-mm; Bio-Rad 170-3622):
(~$120 each)
Combs (Bio-Rad)
Combination comb holder (number 170-3669) (~$110)
10 well comb (170-4326) (~$50)
15 well comb (170-4324) (~$50)
Standard Casting Stand (14 x 13-cm frame and platform; Bio-Rad 170-3689) (~$195)
Wide/Long Combination casting stand (21 x 14-cm frame and platform; Bio-
Rad 170-3704) (~$270)
Leveling Table (for casting gels) (~$230)
Proteinase K powder (Roche Molecular Biochemicals #745 723) (~$82) or
Proteinase K liquid (Amresco, E195-5ml) (~$82)
10X TBE (Sigma, T4415; Bio-Rad 161-770)(~$87 for 4 liters; ~$80 for 5 liter cube)
1M Tris-HCl, pH 8.0 (Invitrogen, 15568-025) (~$38 for 1 liter)
0.5M EDTA, pH 8.0 (Invitrogen, 15575-020; Amresco, E177) (~$48 for 400 ml (4 x 100 ml); ~$27 for 500 ml)
N-Lauroylsarcosine Sodium Salt (Sigma, L9150) (~$34 for 100 g; ~$63 for 250 g)
Ethidium bromide, 10 mg/ml (Bio-Rad 161-0433; Sigma E1510) (~$45 for 10ml; ~$39 for 10 ml)

Restriction Enzymes and appropriate Buffers:

Available from Roche Molecular Biochemicals, New England Biolabs, Promega, or other suppliers
E. coli O157:H7, Salmonella, and Shigella spp:
XbaI Roche: 20,000 units for ~$385
BlnI/AvrII Roche: 1,000 units/New England Biolabs 500 units): ~$350/$300
SpeI Roche: 1,000 units for ~$280
H Buffer (Roche enzymes): 5 ml for ~$26
L. monocytogenes:
AscI (New England Biolabs) 2,000 units for ~$280
ApaI (Roche) 5000 units for ~$65
Clostridium perfringens, Campylobacter jejuni/coli
SmaI (Roche) 5,000 units for ~$205
Campylobacter jejuni/coli
KpnI (Roche) 10,000 units for ~$160
Vibrio cholerae/V. parahaemolyticus (the enzymes below are being considered for PFGE analysis)
NotI (Roche) 1,000 units for ~$281
SfiI (Roche) 1,250 units for ~$105
Other miscellaneous items: ~$5,000 to $8,000 (for spatulas, weighing paper, paper towels, gloves, scalpels/blades, glassware, staining dishes, test tubes, pipets and tips, culture media, etc).

Use of trade names and commercial sources is for identification only and does not imply endorsement by TotalLab.

Equipment & Reagents Required for Pulsed Field Gel Electrophoresis (PFGE)