Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Generating Yeast One-Hybrid DNA-Bait Strains

(Protocol summary only for purposes of this preview site)

Generating DNA-bait strains for Gateway-compatible Y1H screens involves three steps (Fig. 2A in the chapter introduction). The first is to generate an Entry clone containing the DNA-bait of interest. Gateway cloning is used to clone larger baits, such as promoters, into pDONR-P4-P1R. (An alternative set of steps is also presented in this protocol that describes the creation of Entry clones by annealing primers and performing conventional ligation into pMW5a strategy best suited for smaller DNA-baits up to 100 bp.) The second is to transfer this DNA-bait from the Entry clone to the two Y1H reporter Destination vectors, pMW2 (HIS3) and pMW3 (LacZ). A two-step process is used because Entry clones generate a versatile resource that can be used for transfer of DNA-baits into a variety of vectors, for instance, upstream of the green fluorescent proteinencoding ORF to study spatiotemporal expression patterns (Dupuy et al. 2004; Reece-Hoyes et al. 2007; Martinez et al. 2008). The final step is to integrate the HIS3 and LacZ reporter constructs into the genome of the Y1H yeast strain, YM4271. The entire process takes 2432 d, plus sequence confirmation if necessary.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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