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Enter-net/Salm-gene Proposed Protocol for PFGE

Materials

• Cell Suspension Buffer (CSB), (100 mM Tris,100 mM EDTA, pH 8.0)
• Agarose (Biorad Chromosomal)
• TE buffer (10 mM Tris, 1.0 mM EDTA pH 8.0)
• 1% SDS
• Proteinase K
• Lysis buffer (50 mM Tris, 50 mM EDTA, 1 % Sarkosyl, 0.1 mg/ml Proteinase K, pH 8.0)
• XbaI restriction enzyme
• TBE buffer (50mM Tris, 50mM boric acid, 0.5mM EDTA)
• Ethidium bromide
• Equipment for gel electrophoresis of products

Method

Cell preparation

Cells grown overnight on TSA plates or broth. Cells are taken directly from plates or after centrifugation and washing from the sediments and resuspended in cell suspension buffer (CSB), (100 mM Tris, 100 mM EDTA, pH 8.0).

Cell density for plug preparation

0.38 -0.44 at 450 nm (McFarland No. 5).

Preparation of agarose plugs

Prepare 1.6 % agarose (InCert) or 2.0% agarose (Biorad Chromosomal)* in TE buffer (10 mM Tris, 1.0 mM EDTA pH 8.0) cooled down to 50 oC (add 1% of SDS in the final concentration - optional). Add Proteinase K (0.5 mg/ml final concentration) to cell suspension, mix cell suspension 1:1 with agarose and dispense into wells of plug moulds and leave to set.

Lysis of cells in agarose plugs

When set, place plugs in lysis buffer (50 mM Tris, 50 mM EDTA, 1 % Sarkosyl, 0.1 mg/ml Proteinase K, pH 8.0) and incubate for 2 hours (minimum) in a shaking waterbath (54 oC) or 4 hours static (54 oC)

Washing of agarose plugs

Washing of plugs is carried out at 50 oC in a shaking waterbath. Wash twice in sterile distilled water, minimal volume is 5 ml, then wash 2 to 3 times, minimal volume is 5 ml, in TE buffer. Plugs can be stored in TE buffer, 4 oC for several months to years for further application.

Restriction endonuclease digestion in agarose plugs

Place a 3 mm plug in 50-100 µl reaction buffer. Add 0.2 – 0.8 U/µl XbaI and incubate at 37 oC , for 4 hours (minimum).

Preparation of agarose gel

Prepare a 1.0 % (e. g. 100 ml for a 13 X 14 cm gel) or 1.2 % (e. g. 120 ml for 14 X 21 cm gel) agarose (BioRad, Pulsed Field Cert.) gel, respectively, with 0.5 x TBE buffer and load plugs and seal the wells.

Running conditions

2.0 litres or 2.5 litres (for CHEF DR II and CHEF DR III respectively) of 0.5 x TBE buffer (50mM Tris, 50mM boric acid, 0.5mM EDTA)

Molecular reference and internal quality control

As molecular reference marker strain use the CDC (PulseNet) S. enterica serovar Braenderup H9812, which gives rise to a broad spectrum of precisely defined bands. For 20 and 15 lane gels, use a minimum of 3 reference lanes, e.g. lanes 1, 11, 20 or lanes 1, 8, 15. For the EQA gels, use 4 reference lanes e.g. Lanes 1, 8, 14, 20.

We propose to initially standardise PFGE with the following conditions:

• 6 V/cm (200V)
• Switch time 2s-64s
• Agarose gel: 1.2% agarose (21cm long), 1.0% agarose (14cm long)
• Temperature: 14oC
• Run time: 22h (CHEF DR II), 20h (CHEF DR III), 18h (CHEF MAPPER)