I'm going to perform a Y1H experiment. I'm already experienced in Y2H experiments but now I would like to test for DNA-binding capacity in a Y1H system. I'm planning to do this:
* Cloning the Transcription Factors (TFs) into pGADT7 (a Gateway version I have)
* Cloning the DNA promoter into pMW#3 (Gateway version of the Clontech pLacZi vector):
* co-Transformation into YM4271 yeast strain.
* Select cotransformed on -Leu and -Ura plates.
* Check beta-Galactosidase
I wonder if I'm doing it right?
I think that is a good plan, you just need to include your control with the vector harboring the regulatory DNA sequence and without the vector containing the transcription factor (or use empty vector), just to make sure your promoter is not binding some factor(s) from yeast that could give you a high background of beta-gal.
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