When you clone a gene into pGBKT7 and have confirmed that it is the exact sequence you think it is, the next thing you’ll want to do is transform it into yeast. We have a kit for that and the manual is here: PT1172-1.
Some immediate needs are as follows:
- Make a glycerol stock. Just like bacterial freezing media, yeast freezing media consists of 50% glycerol in the typical
- Test whether the gene causes autoactivation of the Y2H system (see if it grows on yeast media lacking tryptophan (confirms that the plasmid is in the yeast) as well as histidine and adenine (tests whether the gene product alone can lead to reporter gene expression). Growth under these conditions means that you can’t use Y2H for your particular TRIM protein.
Next set of experiments:
- Perform a Y2H screen by mating the yeast with your gene in pGBKT7 with the yeast containing a library of human gene cDNA.
- Perform pairwise Y2H assays by mating the yeast with your gene in pGBKT7 with the yeast containing other TRIM and MAGE genes in pGADT7.