1. Subculture strains onto agar media. Grow under appropriate conditions for 1-3 days.

Provide bacterial growth for isolation of DNA for typing

1. Harvest cells and suspend directly into 2ml of cold Pett IV buffer:

Keep samples on ice. Adjust cell densitites to read in the range of Macfarland 6-7.

Important: see footnote 1

Prepare initial cell suspension from which DNA will be isolated. Cold buffer restricts premature cell lysis and subsequent DNA degradation.

Standard cell OD ensures that each sample contains approximately the same amount of DNA

2. Melt agarose (1% chromosomal grade agarose in distilled water) and cool to 56E C in water bath.

20ml DW

Prepares agarose for making bacterial DNA plugs.

3. Use a Gilson pipette to add 0.7ml warm, molten chromosomal grade agarose suspension to 300Fl of bacterial suspension. Mix together.and dispense into 5 chambers of the mould. Set by placing in 'fridge for ca. 10 min.

Note: other methods of pouring agar plugs, such as using plastic syringes as moulds, can be used at the discretion of the laboratory

Preparation of agar plugs containing whole bacterial cells. This stage of sample preparation is a key step for PFGE analysis of chromosomal DNA (see 8).

4. Remove agar blocks by inserting a blunt-tipped glass pipette and place each set of 5 into a 10ml plastic tube containing 3ml ESP buffer:

Add 1mg/ml Proteinase K warmed at 56EC/15 min. Just before use.

Incubate overnight at 56EC.

Note 2: alternative lysing procedures may be used at the discretion of the laboratory.

Lysing of bacterial cells. This leaves only DNA embedded in the solid agar matrix. The DNA will not shear when handled further and remains stable for analysis by electrophoresis.

1. Decant ESP buffer from samples and wash six times, each for at least 20 min periods, in 2 ml of 10:1 TE (10mM Tris-1mM EDTA) buffer. Store blocks in ca. 1.5ml fresh TE buffer in 'fridge. The DNA sample blocks are now ready for PFGE analysis.

Removes excess enzymes and chemicals that would interfere with restriction enzyme analysis.

2. Digestion of bacterial DNA:

Into a standard 1ml eppendorf pipette 100Fl of enzyme buffer for enzyme SmaI. The buffer should be as recommended by the manufacturer. For Amersham, thsi is 80Fl Milli Q, 10Fl Buffer 'T' [1at 0X concn.], and 10Fl BSA)

Prepare buffer for digestion of bacterial DNA with restriction enzymes.

3. Wearing rubber gloves,carefully remove an agar/DNA block using a glass rod. Cut a sliver ca. 1mm wide using a clean cover slip (ensure both edges are straight & parallel). Use the glass rod to place sample into sample buffer and incubate for 1hr at room temp.

Prepare DNA-containing agar slice for restriction enzyme analysis. Incubation in buffer equilibrates sample and makes DNA more amenable to digestion.

4. Remove buffer with a pipette. Add 50Fl of buffer which contains 20 units of enzyme/sample:

For Amersham: SmaI: 2.5Fl Sma1, 5Fl Buffer T, 5Fl BSA, 37.5Fl Milli Q

Incubate at relevant temperature (25-30EC for Sma1) for 5 h.

Note: DNA can alternatively be digested with SmaI overnight, at the discretion of the laboratory. If so, pre-incubation with reaction buffer is not necessary.

Digestion of bacterial DNA with restriction enzyme.

Electrophoresis

1. Prepare 2.5 litres of 0.5 strength TBE buffer using 125ml 10XTBE () and 2,375ml Milli 'Q').

10X Tris-borate EDTA (TBE) buffer:

108g Tris base (0.9 M)

55g boric acid (0.9 M)

40 ml 0.5 M EDTA, pH 8.0

Add Milli ´Q´grade water up to 1 L. Sterilise by autoclaving. Discard if precipitate is observed.

Note: If stored in cold room, allow to stand at room temperature for ca. 1 h before adding to electrophoresis chamber. This prevents accidental freezing of the cooling pipe.

Preparation of electrolyte buffer for electrophoresis of samples.

2. Prepare 1.4% agarose gel (1.0% if using SeaKem Gold: see Footnote 2) for separation of DNA fragments. Measure 5.0ml 10XTBE (Bio-Rad) and make up to 100ml with Milli 'Q'. Suspend 1.4g of separation grade agarose (e.g. Bio-Rad) in this and heat to boiling in microwave. Cool in water bath to 50-56EC.

5-6. Preparation of agarose gel for electrophoretic separation of bacterial DNA fragments.

3. Assemble gel mould using pre-cleaned components and pour gel into mould. The 23 (Pharmacia equipment)- or 30 (Bio-Rad equipment) well-forming comb must be inserted into the assembly before the agarose is poured in!

4. Remove gel well-forming comb and load 22 DNA slices into wells, IN THE ARRANGEMENT DESCRIBED BELOW. Place slice onto the side of a clean plastic scalpel blade or cover slip and gently manipulate into well, ensuring that each slice is straight and adheres onto the front side of the well.

- when using the Bio-Rad 30-well comb, the first and last 4 wells will not be used.

Loading of bacterial DNA sample for electrophoretic separation of restriction fragments. The arrangement of reference and molecular weight standards has been selected to facilitate computer-assisted comparative analysis.

5. Gently overlay wells with molten 1% agarose.

Seals samples into wells so that they remain in place during electrophoresis.

6. Add running buffer (0.5 X TBE) to electrophoresis chamber and run through pump and cooling unit (12EC) ca. 2 hour before use. Place gel (still on perspex unit) into horizontal electrophoresis tank such that samples are nearest the pump inlet. Close cover into place and set electrophoresis conditions on power pack and computer.

Phase 1: 0.5-40s switch time, 22.5 h.

Electrophoretic separation of DNA fragments.

DAY 5

1. Switch off electrophoresis unit. Remove gel from base and gently place into ethidium bromide (3Fl/ml: i.e. 300Fl 10mg standard solution in 1L)). Stain for 7 min.: destain in water for 20 min. View under UV light & photograph.

Printed hard copies / photographs of the final gel should adhere to the following specifications to facilitate computerised analysis:

1. The top of the MW marker should always be visible.
2. B. The gel image should be taken such that the outer edges of the first and last samples are 8.7-9.0 cm apart in the printed image.
3. The normalisation standard pattern (mixture of CNET 068/112) should contain 17 bands of the following approximate sizes (kb): 650, 500, 375, 325, 275, 200, 180, 140, 130, 100, 90, 60, 45, 30, 20, 10, 5. The bands between ca. 5-45kb may be weak but they should be visible.
4. The 650 kb and 5 kb fragments (i.e. the upper and lower bands) in standard CNET 068/112, lane 11 should be 6.0 cm apart ( 5% deviation).

Note: it may be necessary to adjust the running voltage to obtain the above separation levels. Most electrophoresis units use 6V/cm as standard. This should be increased or decreased as required to obtain the results given for the normalisation standard CNET 068/112. Also see Footnote 2.

Visualisation of PFGE-DNA profile.

2. Pump old buffer out from electrophoresis chamber. Rinse with ca. 2.5 L Milli 'Q'. Drain, wipe with an ethanol-soaked cloth and wipe dry.

Cleaning of apparatus. Clean equipment is good equipment!

1. Harvest cells and suspend directly into 2ml of cold Pett IV buffer:

Keep samples on ice. Adjust cell densitites to read in the range of Macfarland 6-7.

Prepare initial cell suspension from which DNA will be isolated. Cold buffer restricts premature cell lysis and subsequent DNA degradation.

Standard cell OD ensures that each sample contains approximately the same amount of DNA

2. Aliquot 1ml suspension into an Eppendorf tube and add 100Fl 37% formaldehyde solution. Incubate at room temperature for 1 h.

Formaldehyde deactivates bacterial DNAse activity that may occur in some strains. If left untreated sample DNA degrades and cannot be analysed.

3. Centrifuge the suspension for 10 min. at high speed. Wash three times in 1ml Pett IV buffer.

Remove formaldehyde, which would interfere with subsequent procedures.

4. Resuspend washed cell pellet in 600Fl Pett IV buffer.

Prepare bacterial cells for embedding in agarose (see 7).

5. Warm bacterial suspension to 37EC by placing in water bath for a few minutes.

Prevents premature solidification of agar plug (see 7).

6. Seal base of agar plug mould with tape.

Prevents leaking of agar/bacterial suspension from sample mould!

7. Melt agarose (1% chromosomal grade agarose in distilled water) and cool to 56E C in water bath.

20ml DW

Prepares agarose for making bacterial DNA plugs.

8. Use a Gilson pipette to add 0.7ml warm, molten chromosomal grade agarose suspension to 300Fl of bacterial suspension. Mix together.and dispense into 5 chambers of the mould. Set by placing in 'fridge for ca. 10 min.

Note: other methods of pouring agar plugs, such as using plastic syringes as moulds, can be used at the discretion of the laboratory

Preparation of agar plugs containing whole bacterial cells. This stage of sample preparation is a key step for PFGE analysis of chromosomal DNA (see 8).

9. Remove agar blocks by inserting a blunt-tipped glass pipette and place each set of 5 into a 10ml plastic tube containing 3ml ESP buffer:

Add 1mg/ml Proteinase K warmed at 56EC/15 min. Just before use.

Incubate overnight at 56EC.

Note 2: alternative lysing procedures may be used at the discretion of the laboratory.

Lysing of bacterial cells. This leaves only DNA embedded in the solid agar matrix. The DNA will not shear when handled further and remains stable for analysis by electrophoresis.