35- Classical epifluorescence microscope equipped with a 100×
objective, appropriate fluorescent filters, fluorescent lamp, and
CCD, EMCCD, or sCMOS camera. - Microscope glass slides and coverslips, Gene Frame and aga-
rose for preparing agar pads.
3 Methods
- Starting from the glycerol stock, grow an overnight culture of
the strain expressing xfp-mutL in a desired medium to satura-
tion at 37 °C. Add the inducer to the growth medium when
necessary (see above). - Dilute 400-fold the saturated culture and incubate at 37 °C
until O.D. 0.15–2.0 is reached (see Notes 7 and 8 ). - Centrifuge 1 mL of the exponentially growing culture for
1 min at 13,000 rpm (12000 × g) to concentrate cells. - Throw away the supernatant, resuspend cells by pipetting and
depose 1–2 μL of suspension on the agarose pads prepared
before (see below). - Dissolve agarose (1.5%) in the medium in which cells have
been grown using a microwave oven (see Note 9). If the auto-
fluorescence of the growth medium is too high, as in the case
of LB, dissolve agarose in the minimal medium or simply in
M9 (see Note 10). If required, supplement the growth medium
with the Plac inducer IPTG. - Take a clean microscope glass slide (dimensions adapted to
your microscope). Attach the Gene Frame, the adhesive system
for easy agarose pad preparation, in the middle of the slide (see
Note 11). - Transfer 100 μL or 200 μL of the warm agarose in the middle of
the Gene Frame, depending on the Gene Frame dimensions, and
cover rapidly with the proper coverslip (see Notes 12 and 13 ). - Leave the slide in a horizontal position for 10 min at the room
temperature to allow the agarose to solidify. - Remove the coverslip and the Gene Frame upper plastic cover.
Plate 1–2 μL of the concentrated exponential phase culture on
the agarose pad and allow the liquid to disperse by turning the
slide in different directions 3–4 times. - Leave to dry for a few minutes at room temperature until no
more liquid is detectable on the agarose pad (see Notes 14
and 15 ). - Put the coverslip, and gently press to assure the proper sealing
of the coverslip to Gene Frame. Try avoiding making air bub-
bles (see Note 13).
2.3 Microscopy
and Mounting
3.1 Growth
3.2 Preparing
Agar Pads
Detecting Mutations in Living Cells