Biomedical Engineering Reference
In-Depth Information
from the pinning (especially using a robot) causes liquid to
bead on the agar surface, disturbing colony growth and
allowing cross-contamination.
7. Cooling YPD to 60 °C before adding G418 or ClonNat mini-
mizes the possibility of thermal inactivation. 100
g/mL G418
is preferred for maintaining Kan
R
yeast deletion arrays because
many Kan
R
traffi cking mutants show impaired growth at
200
ʼ
g/mL G418, the concentration typically used to for
Kan
R
selection.
8. Other non-glucose carbon sources such as galactose can be
used (at fi nal concentration of 2 %).
9. Each batch of glucose oxidase may have differing unit activity/
gram.
10. Undigested plasmid results in background colonies that con-
tain the intact plasmid and
SUC2
locus. Therefore, testing iso-
lates to make sure they do not grow on SD-URA is essential
because the
URA
3 is contained on the backbone plasmid and
not in the insert.
11. For a high-effi ciency yeast transformation protocol,
see
Gietz
and Woods [
10
].
12. This can be done on 100 mm petri plates, rather than
OmniTrays, to reduce cost.
13. Yeast arrays frozen in 96-well plates are typically covered with
cryogenic sealing tape that should be removed while plates are
still frozen to prevent cross-contamination. Cover plates with
lid and allow them to thaw at room temperature (approxi-
mately 15 min) before pinning. After pinning, reseal plates
with fresh sealing tape and freeze. Yeast can survive several (at
least three) freeze-thaw cycles without signifi cant loss of
viability.
14. Further condensing the 384 arrays to create 1,536 arrays
results in more uniform colony size and reduces variability.
15. For better measurements we typically randomize positions
around the plate to account for edge effects or nonuniform
distribution of reagents.
16. We use the following depths when making frozen stocks, since
the yeast can ride up the pins: 13, 15, and 16 mm for water
reservoirs, 14 mm for 10 % bleach reservoir, and 17 mm for
100 % ethanol reservoir. When working with 96-density agar
arrays, these depths can be reduced considerably, since the
yeast is on the bottom of the pins. Any water reaching the pin
support plate would prevent pins from fl oating freely. Thus it
is important to reduce wash station depth where possible.
17. The wash reservoirs are set up to submerge pins in incremental
depths in each step. For example, the fi rst wash should sub-
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