Materials and reagents

 
1 TSA (Tryptone 15 g, Soy peptone 5 g, NaCl 5 g, Agar 15 g/L, pH 7.3) or LB plates (Sambrook et al. 1989).

2 10 µl disposable inoculation loops (Simport, L200-2)

3 0.5 M EDTA pH 8.0.

4 100x TE buffer (1 M Tris, 0.1 M EDTA, pH 8.0).

5 TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

6 Guanidine thiocyanate -EDTA-Sarkosyl (GES) solution.

  per 100 ml: 60 g guanidine thiocyanate (Sigma, #G-9277)
  20 ml 0.5 M EDTA pH 8.0.
  20 ml sterile water.

Facilitate dissolving of all components by heating to 65ºC; cool down the solution and add 1 g N-lauroyl sarkosine ( Sigma, # L-5125). Adjust to 100 ml with water and filter sterilize using a 0.45 µm filter; store at RT.

Comment: Guanidine thiocyanate is harmful, wear suitable protective clothing.

7 Resuspension Buffer (RB; 0.15 M NaCl, 0.01 M EDTA pH 8.0).

8 Ammonium acetate (7.5 M) per liter: 578.1 g NH4Ac in 1000 ml water.

9  Chloroform/iso-amyl-alcohol 24/1 (v/v)
Comment: Chloroform/iso-amyl-alcohol is poisonous; wear suitable protective clothing and use in a well ventilated area, such as a fumehood.

10 Ethanol 70 % (non denatured)

11 RNAse solution (2.5 mg/ml)  

   per 20 ml:  50 mg RNAse

   20 ml water

   Incubate 10 minutes at 1000C

   Aliquot and store at -200C.

   Dilute 10 x before use.
Comment: All solutions and media except for those described in 6, 9, and 10, need to be sterilized by autoclaving.

Experimental protocol

1. Use as starting material a well-grown 24 - 48 h old pure culture from an agar plate (TSA or LB).

2. Remove cells with a sterile 10 l loop, taking one loop full, spherical on one side and flat on the other side and resuspend in 500-1000 µl RB.

3. Pellet cells by centrifugation and remove the supernatant using a 1000 µl tip.

4. Pellet cells again and remove the remaining supernatant using a 200 µl tip.

5. Add 100 µl TE, pH 8 and mix using a pipette.

Comments: For isolates that produce excess polysaccharides such as several xanthomonads, use 1000 µl RB or repeat wash with RB and centrifuge for 10 minutes or even longer. An enzyme treatment can be added for microbes that are difficult to lyse. 10 mg lysozyme per ml TE can be added in step 5, incubation for 30 minutes at 37ºC. Alternatively achromopeptidase, mutanolisine or pronase may be used (Versalovic et al. 1994).

6. Add 500 µl GES and mix the reaction vials gently.

7. Incubate the mixture 5 min. on ice.

8. Add 250 µl cold (-200C) ammonium acetate (7.5 M), mix the tubes by shaking the tubes gently, incubate them 5 min. on ice.

9. Add 500 µl chloroform/iso-amyl-alcohol (24/1), shake vigorously until the solution is homogeneously milky, and centrifuge the mixture 10 min. or until the upper phase is clear.

10. Prepare numbered tubes with 378 µl iso-propanol at -200C.

11. Carefully remove 700 µl DNA-solution of the upper phase using a 1000 µl tip and add the DNA solution to the tubes containing iso-propanol.

Comments: To facilitate removing the upper phase without including some of the interface material, and to prevent shearing of the DNA, the end of the tips used should be cut off.

12. Shake gently until a white cloud of precipitated DNA becomes visible.

Comment: The reaction vials can now be stored at -200C.

13. Pellet DNA by centrifugation and remove the supernatant as described in step 3 and 4.

14. Add 150 µl 70 % ethanol but do not mix, centrifuge briefly and remove ethanol with 200 µl pipette, repeat centrifugation and remove residual ethanol.

15. Air dry the DNA pellet and redissolve in 200 µl TE, pH 8.

16. Incubate overnight at room temperature or at 40C, alternatively 4 hours at 450C until the DNA is dissolved, add 25 µl RNAse (250 µg/ml) and mix gently.

17. Incubate 1 hour at 37°C and store the DNA at 40C or at -200C.

18. Determine the DNA concentration using a spectrophotometer at 260 nm (1 OD260 = 50 g/ml) and adjust it to 50 ng/µl.


Rep-PCR fingerprinting protocol.

Often a detailed characterization of strains can be obtained by applying one primer (set). The BOX primer is generally recommended since it generates robust fingerprints, and yields a highly complex fragment pattern. The REP primer set generates a lower level of complexity, but still yields reproducible and differentiating fingerprints (see Fig. 7). The ERIC primer set is more sensitive to sub-optimal PCR conditions, such as the presence of contaminants in the DNA preparations but also generates highly discriminatory patterns (see Fig. 3A). A small pilot experiment is usually carried out to find the optimum primer set for a given application.

- PCR amplification reactions.

PCR reactions and stock solutions are prepared on the bench. Filtertips are used to prepare stock solutions only. Wearing gloves is not essential. Care is taken that everything is efficiently organized to ensure a swift preparation of the reactions, and to avoid DNA contamination.

Materials and reagents

1 5 x Gitschier Buffer (Kogan et al. 1987)

 -Prepare the following stock solutions and autoclave them separately:

  1 M (NH4)2SO4
  1 M Tris-HCl (pH 8.8)
  1 M MgCl
  0.5 M EDTA (pH 8.8)

 -To prepare 200 ml of 5 x Gitschier combine;
  16.6 ml of 1M (NH4)2SO4
  67 ml of 1 M Tris-HCl (pH 8.8)
  6.7 ml of 1 M MgCl2
  1.3 ml of a 1: 100 dilution of 0.5 M EDTA (pH 8.8)
  2.08 ml of a 14.4 M commercial stock of b-mercapto-ethanol, stored at 40C

  Adjust finally to 200 ml with approximately 106 ml water and mix. Store at -200C in 1 ml aliquots.
Comment: BSA is added while preparing master mix.

2 Ultra pure dNTP set, 100 mM each (Pharmacia, #272035-1)
  100 mM solutions are mixed 1:1:1:1 to obtain a solution
  with 25 mM of each nucleotide, the solution is divided in 100 ml aliquots 
  and stored at -20C.

3 BSA, 20 mg/ml nuclease free (Boehringer, #711454), the solution 
  is divided in 20 ml aliquots and stored at -20C.

4 DMSO 100% (Fluka, #41640), the solution is divided in 0.5 ml aliquots and stored at -20C, 
  one working solution is kept at RT.

5 Autoclaved ddH2O, 2.5 ml aliquots in 5 ml screw cap vials are stored at RT.

6 0.3 µg/µl primer 1; BOX A1R, ERIC 1R or REP 1R. 

7 0.3 µg/µl primer 2; ERIC 2 or REP 2I; primer solutions are divided in 200 ml aliquots 
  and stored at -20C.
Primer Sequence Reference
BOX A1R5'-CTACggCAAggCgACgCTgACg-3' Versalovic et al. 1994
ERIC 1R5'-ATgTAAgCTCCTggggATTCAC-3' Versalovic et al. 1991
ERIC 25'-AAgTAAgTgACTggggTgAgCg-3' Versalovic et al. 1991
REP 1R5'-IIIICgICgICATCIggC-3' Versalovic et al. 1991
REP 2I5'-ICgICTTATCIggCCTAC-3' Versalovic et al. 1991

8 Taq DNA Polymerase 5 U/µl (e.g. PE, # N8080070) stored at -20C.

9 Mineral oil (Sigma, # M-3516) stored at RT.

10 Thermal cycler (e.g. Perkin Elmer 480 or MJ Research PTC 100 or 200).
Experimental protocol

1. Number PCR reaction tubes.

  1. Prepare the master mix.



per one 25 µl reaction: for ... 25 µl reaction: Stock solution
5 µl... µl 5 x Gitschier Buffer
0.2 µl... µl BSA, 20 mg/ml
2.5 µl... µl DMSO, 100%
12.65 µl... µl ddH2O, use 13.65 µl for BOX
1.25 µl... µl mix of dNTP's (1:1:1:1)
1 µl... µl primer 1
1 µl... µl primer 2, not applicable for BOX
0.4 µl... µl Taq DNA Polymerase, 5 U/l

3. Mix gently.

4. Collect by centrifugation.

5. Aliquot 24 µl master mix to each tube.

6. To reduce evaporation overlay the mix with a drop of mineral oil.

Comments: Mineral oil can be applied in sample tubes anywhere between step 4 and 8. To prevent cross contamination of the samples it should be applied as early as possible.

7. Add 1 µl of the samples to be fingerprinted to the reaction mix.

8. Collect by centrifugation.

9. Insert samples and control reactions in thermal cycler .

10. Start the PCR with an incubation at 95oC for 7 minutes, time delay file (#2) for PE 480. Continue using 30 cycles or 35 cycles for whole cell rep-PCR, using a step cycle file (#4) consisting of:
BOX
ERIC
REP
94°C for 1 min.
94°C for 1 min.
94°C for 1 min.
53°C for 1 min.
52°C for 1 min.
40°C for 1 min.
65°C for 8 min.
65°C for 8 min.
65°C for 8 min.

11. Terminate the PCR reactions with an extension at 65oC for 16 min. (time delay file #2), and store at 4°C (soak file #1).

Comments: Instead of the Perkin Elmer 480, the Peltier element based thermo-cyclers such as the MJ Research PTC 100 and PTC 200 with a 96 well format and a heated lid can be used. These machines are faster, use smaller tubes with thinner walls, do not "over- or under-shoot"target temperatures and do not need mineral oil. Therefore the following cycles in the "block"mode are used to obtain similar profiles in a shorter time. The PCR is started with an incubation at 95oC for 2 minutes and continued using 30 cycles or 35 cycles, for whole cell rep-PCR, consisting of:
BOX
ERIC
REP
94°C for 3 s,

92°C for 30 s.
94°C for 3 s,

92°C for 30 s.
94°C for 3 s,

92°C for 30 s.
50°C for 1 min.
50°C for 1 min.
40°C for 1 min.
65°C for 8 min.
65°C for 8 min.
65°C for 8 min.

Terminate the PCR reaction with an extension at 65oC for 8 min., and store at 4°C.

12. Store the completed PCR reactions at 4oC and whole cell rep-PCR products preferably at -20oC, to prevent breakdown of the amplified products or use directly for gel electrophoresis, as described below.

- Gel electrophoresis-mediated separation of rep-PCR amplified genomic fragments.

Normally, an agarose gel is sufficient to separate the rep-PCR generated fragments (see Fig. 3A), although polyacrylamide gel electrophoresis may also be used.

Materials and reagents

1 Agarose LE (Seakem, # 50004)

2 50x TAE electrophoresis buffer:

  Add 242 g Tris (free base), 57.1 ml glacial acetic acid, 18.61 g Na2 EDTA and ddH2O up to 1 liter,
  store at RT.

3 Ethidium bromide (10 mg/ml) (Sigma), store at 4°C.

4 6x loading buffer : consists of 0.25% bromophenol

  blue, 0.25% xylene cyanol FF and 15 % Ficoll 400 [all w/v in ddH2O],

  store at -20°C.

5 1 kb DNA size ladder (Gibco BRL, # 15615-016), store at -20°C.

6 Horizontal gel electrophoresis apparatus H4 (Gibco BRL, # 11025012).

7 Gel tray 20.0 x 25.0 cm (7.9 x 9.8 inch) (Gibco BRL).

8 Comb, 30 tooth, 1 mm thick. (Delrin, # 1951CO).

9 Electrophoresis power supply (Gibco BRL, Model 250).

10 UV transilluminator (wavelength 312 nm) (Fotodyne, 3-3000).

11 Face protecting shield! (Fotodyne, model K).

12 Camera and suitable filters (Kodak, MP4).

13 UV-sensitive film (Polaroid, 55 or 57 film).

14 Oven or water bath at 50-70oC.

Next pages 12-17