Primer pairs will amplify sequences present as a single copy in the mouse genome with the Universal Genotyping Protocol. Includes: b-Galactosidase (LacZ); cre-recombinase; CFP; diphtheria toxin; dsRED; Fabpi-200; Fabpi-500; flp recombinase; GFP/BFP/YFP; human growth hormone (complete); human growth hormone (transcriptional stop); luciferase (click-beetle); luciferase (firefly); neomycin phosphotransferase; SRY (male-specific); tTA (tet-on). - [Read PCR Genotyping Primer Pairs Protocols]
Protocol describes how to generate a plasmid construct (pBAIT) that expresses a target protein fused to the bacterial LexA protein. PBAIT is cotransformed into yeast with a lexAop-lacZ reporter plasmid carrying the bacterial lacZ gene under the control of the lexA operator. The recipient yeast strain contains a chromosomally integrated leu2 reporter gene, also under the control of the lexA operator. - [Read Two-hybrid Systems Stage 1: Characterization of a Bait-LexA Fusion Protein Protocol]
In this stage of the protocol, a mammalian cDNA library constructed in a plasmid such as pJG4-5 is transformed into yeast strains containing pBAIT and the lexAop-lacZ reporter plasmid. PJG4-5 expresses the cloned cDNAs from a cassette containing a transcriptional activation domain and other moieties under the control of the yeast GAL1 promoter. - [Read Two-hybrid Systems Stage 2: Selecting an Interactor Protocol]