Using Yeast Two-Hybrid Methods to Investigate Large Numbers of Binary Protein Interactions - Genomics: Essential Methods - page 183

• SD plates − trp − leu − his (+2.5m M 3-AT) (HIS3 reporter selection plates)
• SD plates − trp − leu − ade (ADE2 reporter selection plates)
• Sterile velvets
• Replicating block.
Method
1 Pipette the required amount of sterile water (4 μ l per mating reaction) into the
required number of wells of a 96-well plate.
2 Using a sterile toothpick, pick a small sample of a bait colony and resuspend the yeast
in the required well. It may help to leave the toothpick in the well until finished. This
avoids cross-contamination between samples or wells.
3 When all bait clones are resuspended, mix with tips of a multichannel pipette before
spotting out 3 μ l of each suspension onto a dry YPAD plate (150mm vented plates are
required for standard 96-well format arrays).
4 Allow spots to spread and dry.
5 While bait spots are drying on the YPAD plate, resuspend prey clones as for baits.
6 Pipette 3 μ l of prey clones directly on top of appropriate bait spots on the YPAD
plate.
7 Allow spots to dry; then invert plate and transfer to 30 ◦ C incubator.
8 Allow cells to mate on YPAD plate for 12-16 h at 30 ◦ C.
9 Pre-dry SD ( − trp − leu) diploid selection plates in a sterile hood.
10 Stretch a sterile velvet over a replication block and invert the YPAD plate onto the
velvet.
11 Apply even gentle pressure with finger tips to the back of the plate.
12 Remove YPAD mating plate and replace it with the pre-dried SD ( − trp − leu) diploid
selection plate. Again, apply gentle pressure to the back of the plate. Remove plate
and replace lid.
13 Incubate the SD ( − trp − leu) plate at 30 ◦ Cfor2daysuntilsolidevengrowthis
observed on spots.
14 Invert the SD ( − trp − leu) plate onto a sterile velvet and apply gentle even pressure.
The color of the colonies will change slightly as they are transferred to the velvet. Too
much pressure at this stage will result in too much yeast being transferred and results
will be less clear.
15 Place a thick (75 ml of media per 150 mm plate) − trp − leu diploid selection plate
onto the velvet and apply even gentle pressure. Remove plate and replace lid.
16 Without changing the velvet place an SD ( − trp − leu − ade) plate on to the velvet and
again apply gentle even pressure. Remove plate and replace lid. l
17 Using the same velvet place an SD ( − trp − leu − his + 2.5m M 3-AT) plate on to the
velvet and again apply gentle even pressure. Remove plate and replace lid.