Biomedical Engineering Reference
In-Depth Information
Manual Pinning
1. Prepare a suffi cient number of YPD + G418+ ClonNat plates
and ensure they are clearly labeled.
2. The 96-pin manual replicator must be sterilized immediately
prior to each pinning step to avoid contamination of the arrays.
Set up fi ve reservoirs of increasing depths (volumes of reser-
voirs may vary): three reservoirs of sterile water, one of 10 %
bleach, and one of 100 % EtOH (
see
Note 16
).
3. Place the replicator in each reservoir as follows: 13 mm water
reservoir (1 min), 10 % bleach (20 s), 15 mm water reservoir
(5 s), and 16 mm water reservoir (5 s). Finally, place the rep-
licator in the 100 % ethanol reservoir (5 s). Allow the excess
ethanol to drip from the pins, and then ignite with a fl ame
while holding the replicator in a horizontal position. Allow
the replicator to cool before contacting the colonies (
see
Notes 17
and
18
).
4. Pin by gently lowering the pinhead onto the yeast. Then visit
the destination plate and repeat (
see
Note 19
).
(a) For every four 96-density arrays (or visit one plate four
times if four replicates are desired) to make a single
384-density array with a sterile 96-pin replicator, use align-
ment holes A through D on the LIBRARY COPIER™ for
successive pinnings.
(b) For pinning from liquid, lower the replicator pin head into
the bottom of the well and gently agitate to ensure yeast
are well mixed.
5. If desired, add appropriate control strains to empty positions
of each 384-array plate by hand (
see
Note 20
).
6. Incubate 384-density array plates for 2 days at 30 °C and store
at 4 °C for up to 2 months.
7. Test arrays must be replicated to fresh media and grown for 2
days before use in phenotypic screens (
see
Note 21
). Replicate
the 384-density array onto fresh YPD + G418 + ClonNat plates
using a sterile 384-pin replicator. Place the LIBRARY
COPIER™ over an open OmniTray with the pair of nine-
alignment holes on the front of the frame. Use the middle hole
(E) to align the replicator with the yeast colonies.
8. Incubate the plates for 2 days at 30 °C. These 384-density
“test” arrays are now ready for use in phenotypic screens.
Freezing an Array
1. Prepare selective yeast medium with 15 % (V/V) glycerol and
add 150
ʼ
L per well to the desired number of 96-well microti-
ter plates.
