E. coli Source Tracking by Rep-PCR DNA Fingerprinting
 
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Methods: Box Fingerprint Protocol (9-19-00)

Note: this protocol has been changed from our publication (Appl. Envirn. Microbiol. 66:2572-2577) whole cells from single colonies are now used instead of whole cells from overnight liquid cultures

BoxA1R primer sequence (5' CTA CGG CAA GGC GAC GCT GAC G 3'). Versalovic, J., F. J. deBruijn, and J. R. Lupski (1998)

1. Prepare whole cell template(s)

  • Streak isolate(s) onto plate count agar (Difco)
  • The following day swirl an overnight colony (colonies) into 100 µl sterile water using a 1 µl loop

2. Prepare PCR master mix as follows:

  • for each reaction

diH2O

12.35 µl

5X Gitschier buffer*

5

DMSO

2.5

Box primer

1.3 (0.3 µg total, exact volume depends upon primer conc.)

dNTPs

1.25 (Pharmacia set, 100 mM each mixed 1:1:1:1)

BSA

0.2 (Boehringer, 20 mg/ml nuclease free, #711454)

Taq

0.4 (we use Pomega Taq DNA polymerase in storageBuffer B)

Total

23
  • Divide the master mix into 23 µl aliquots
  • Add 2 µl of whole cells in water for each isolate
  • Add 2 µl of water to one aliquot of reaction mix for a no template control reaction (A no template control should be included in each set of reactions)

3. PCR conditions: we use an MJ Research PTC 100 with a 96 well format

  • 95°C for 2 minutes
  • 30 cycles
    • 94°C for 3 seconds
    • 92°C for 30 seconds
    • 50°C for 1 minute
    • 65°C for 8 minutes
  • 65 C for 8 minutes
  • Hold at 4 C
  • Store PCR reactions frozen at -20°C until use

PCR protocols from Rademaker and de Bruijn (1997)

Please note: the next section was revised on 3-04-02

*5X Gitschier Buffer

As found in:

Rademaker, J.L.W., F.J. Louws, and F.J. de Bruijn. 1998. Characterization of the diversity of ecologically important microbes by rep-PCR genomic fingerprinting. Molecular Microbial Ecology Manual 3.4.3:1-27.

Prepare and autoclave stock solutions of each reagent, and subsequently combine them to prepare a 5X buffer. For 200 ml of 5X buffer, proportion the stock solutions to achieve a final concentration of each of the following reagents using sterile, double-distilled water:

To prepare 200 ml of 5X Gitschier combine:
Final concentration

16.6 ml of a 1 M (NH4)2SO4

83 mM (NH4)2SO4

67 ml of a 1 M Tris-HCl pH 8.8

335 mM Tris-HCl pH 8.8

6.7 ml of a 1 M MgCl2

33.5 mM MgCl2

1.3 ml of 1:100 dilution of a 0.5 M EDTA

33.5 µM EDTA

2.08 ml of a 14.4 M commercial stock of ß-mercapto-ethanol

150 mM ß-mercapto-ethanol

Adjust final volume to 200 ml with approximately 106 ml water.

Dispense buffer into sterile 1.5 ml microcentrifuge tubes and store at -20° C. This buffer may be stored for several months.