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.
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.
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
- 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.
|BOX A1R||5'-CTACggCAAggCgACgCTgACg-3'||Versalovic et al. 1994|
|ERIC 1R||5'-ATgTAAgCTCCTggggATTCAC-3'||Versalovic et al. 1991|
|ERIC 2||5'-AAgTAAgTgACTggggTgAgCg-3'||Versalovic et al. 1991|
|REP 1R||5'-IIIICgICgICATCIggC-3'||Versalovic et al. 1991|
|REP 2I||5'-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.
|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
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
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:
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
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.