Biomedical Engineering Reference
In-Depth Information
Fig. 3
Invertase overlay assay: a portion of a 1,536-colony array is shown before and after addition of the
overlay reagents. Color development proceeds during a 20-35 min incubation period. Wild-type yeast become
honey brown
, while endocytosis mutants turn
darker brown
and recycling mutants remain
white
1. Pin the array to be assayed onto YEPF OmniTrays
(in 1536-density format if possible). Incubate at 30 °C for
24 h (
see
Notes 21
and
22
).
2. To prevent washing away nearby colonies when pouring the
agar overlay, cut a trough in the agar along the narrow side of
the OmniTray between the plate rim and the fi rst row of colo-
nies (
see
Note 23
).
3. Capture an image of the plates prior to performing the inver-
tase assay using an imaging system or a fl atbed scanner. This
will provide a measure of colony growth (
see
Note 24
).
4. If using refrigerated reagent stocks, allow them to equilibrate
to room temperature before starting the assay.
5. Microwave the 4 % agar until it is near boiling. Add a stir bar
and place the hot agar onto a hot plate set to ~200 °C
(
see
Note 25
).
6. Add 26 mL dH
2
O to the 50-mL tube containing 1.72 g ultra-
pure sucrose. Dissolve the sucrose by inverting the tube.
7. Add 4 mL 1 M NaOAc pH 5.5 and 0.8 mL NEM to the Falcon
tube.
8. Add 0.4 mL HRP 1 mg/mL, 0.32 mL 1,000 U/mL glucose
oxidase, and 2.4 mL 10 mg/mL
o
-dianisidine and invert twice.
9. Add 7 mL hot 4 % agar to the tube and invert twice. Pipette 20 mL
of the reagent and agar mix rapidly into each of two OmniTrays by
directing the fl ow into the middle of the cut trough.
3.2.1 Invertase
Overlay Assay
10. Record the start time and allow 20-35 min reaction time
before imaging. Leave lids off the plates to avoid heat-related
artifacts (
see
Notes 26
and
27
).
11. Repeat for the remaining arrays.
