notice technique - Biocentric

1. Inoculate MacConkey bouillon with a pea-sized amount of feces or, when feces
is fluid, by using a 10 µl inoculation loop and incubate overnight at 35 +/-2°C.
2. Plate bouillon with a 10 µl inoculation loop on a MacConkey agar plate and incubate
overnight at 35 +/-2°C.
3a. Remove colonies from the plate using a sterile cotton swab which was soaked
before in water (molecular biology grade). Elute swab in a tube filled with 500 µl
of water (see above).
3b.Wash colonies from the plate using 1.5 ml sterile 0.9% saline solution, transfer
to a tube and mix well. Spin in a table-top centrifuge with an aerosol-tight rotor
in a class II safety cabinet for 5 min at 8000 x g and discard supernatant. Resuspend
pellet in 300–500 µl of water (molecular biology grade) and vortex.
4. Incubate suspension from 3a or 3b for 15 min at 95°C in a heating block.
5. Spin down for 5 min at maximum speed in a standard table-top centrifuge and
transfer supernatant to a new tube. Use 5 µl of the lysate for amplification (dilute
if necessary).
Amplification
Two separate PCR reactions have to be performed with each sample: one with PNM
EHEC 1 and one with PNM EHEC 2. Prepare the amplification mixture (45 µl) in a
DNA-free room. The DNA sample should be added in a separated area.
Per tube mix:
– 35 µl PNM EHEC 1 or PNM EHEC 2
– 5 µl 10x polymerase incubation buffer – not provided
– x µl MgCl 2
solution 1) – not provided
– 1–2 unit(s) thermostable DNA polymerase (refer to manual) – not provided
– y µl water to obtain a volume of 45 µl (not considering volume of enzyme) – not
provided
– Add 5 µl freshly isolated DNA solution (20–100 ng DNA) leading to a final volume
of 50 µl (not considering volume of enzyme).
1)
Depending on the enzyme/buffer system used, the optimal MgCl 2
concentration
may vary between 1.5 and 2.5 mM. Please note that some incubation buffers already
contain MgCl 2
.
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