METHODS: REP-PCR Overview (10-25-00)

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

This protocol is written for high-throughput analysis: for the set-up of 54 PCR reactions which are then run on 2 gels, it may be modified as needed

Day 1

Streak 53 isolates of E. coli onto plate count agar (Difco) for single colony isolation, streak 4 isolates per plate incubate overnight at 37^oC

Day 2

1. label top of sterile 96-well microtiter plate with isolate numbers

2. using repeat pipettor, pipet 100 µl of sterile water into each labeled well

3. using 1 µl blue plastic loop pick a colony off plate for each isolate and swirl into water in 96-well plate

4. set plate with colonies in water aside, defrost PCR reagents, mix master mix (on ice) and aliquot 23 µl of master mix into individual wells of a low profile multiplate (MJ Research), the multiplate can be easily cut with scissors (see Box fingerprint protocol for additional detail)

Master Mix
56 reactions (two sets of 27 plus 2 additional reactions)

Add 22.4 µl of taq (Promega taq polymerase Buffer B)

5. pipet 2 µl of colony swirled in water into appropriate aliquot of PCR mix, mix with yellow tip, keep PCR aliquots on ice, check off each isolate on microtiter plate lid as it's added to a PCR reaction mix, add 2 µl water to no template control

6. seal reactions with microseal film (MJ Research) and start PCR (see Box fingerprint protocol for PCR conditions) PCR can be run overnight or set up early in the morning in which case the reactions can be run on gels later in the afternoon

Day 3

Run PCR reactions on gels overnight (see running and staining Box fingerprint gels protocol)

Day 4

Stain gels and enter digital images into BioNumerics database (see adding gel images to the BioNumerics database protocol). The source(s) of the E. coli isolated from water may now be identified by comparison of these fingerprint images to the fingerprints from known sources in the database (see identifying the source groups of water isolates using the database). The same database contains isolates from both known and unknown sources, so that comparisons can be easily made.