Biology Reference
In-Depth Information
DNA-DB/bait
(pBGKT7)
Marker:
AD/cDNA library
(pGADT7)
Marker:
TRP1
LEU2
CM
Frecuency of co-
transformation
IM-LS
IM-LS
A B C D
A B C D
A B C D
A B C D
A B C D
CM
IM-LS
IM-MS
IM-HS
Fig. 2
Screening a cDNA library. A yeast culture is co-transformed with DB-bait and AD-cDNA library. Dilutions
of the co-transformed culture are inoculated in CM media to determine the frequency of co-transformation, the
rest of the culture is plated in IM-LS media to select for interactors in low stringency conditions. Colonies
grown in IM-LS are replicated to a new plate of IM-LS (the diagram represents four positive clones labelled as
A, B, C and D). The yeast are collected from one streak using a sterile toothpick and resuspended in 100 μl of
deionized sterile water. Add 5 μl of each suspension in plates of CM (as a growth control), and IM-LS, IM-MS
and IM-HS to determine the strength of the interaction. Examples of growth level expected from strong (clone
A), medium (clone B), weak (clone C) and very weak (D) interactions are shown
