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Latest Questions
How to narrow down the probe sequence selection range?
The probe design range can be determined by analyzing the promoter sequence. If the range is too broad, initially clone 0.5-1kb fragments into a luciferase reporter vector to verify promoter activity and whether it is regulated by the transcription factor. The core promoter can be identified through serial truncation validation. If the transcription factor under study has a known binding motif, the probe can be selected around that motif site. Alternatively, after performing ChIP or DNA pull-down to find enriched sequences, design segmented probes for validation.
What is the experimental workflow for Nuclear Yeast library construction and screening?
1、Library Construction:
Total RNA extraction → mRNA isolation & purification → Double-stranded cDNA synthesis & purification → In vitro ligation of ds cDNA into pGADT7 vector → E. coli library culture → Library plasmids → Transformation into Y187 yeast host → Y187 yeast library culture.
2、Two-Hybrid Screening:
(1) Mating method:
Bait recombinant pGBKT7 plasmid construction → Autoactivation test: Co-transformation of bait recombinant plasmid & empty pGADT7 into Y2HGold yeast host → Mixing Y2HGold yeast culture containing bait with Y187 yeast library culture → Plating on selective plates based on autoactivation results → Picking positive colonies → Culturing positive yeast & plasmid extraction → PCR identification of positives → Sending for sequencing → Sequencing result analysis.
(2) Co-transformation method:
Bait recombinant pGBKT7 plasmid construction → Autoactivation test: Co-transformation of bait recombinant plasmid & empty pGADT7 into Y2HGold yeast host → Transformation of library plasmids into Y2HGold competent cells containing bait → Plating on selective plates based on autoactivation results → Picking positive colonies → Culturing positive yeast & plasmid extraction → PCR identification of positives → Sending for sequencing → Sequencing result analysis.
3、One-Hybrid Screening:
Bait recombinant pABAi plasmid construction → Autoactivation test: Transformation of linearized bait recombinant plasmid & empty pGADT7 into Y1HGold yeast host → Transformation of library plasmids into Y1HGold competent cells containing bait → Plating on selective plates based on autoactivation results → Picking positive colonies → Culturing positive yeast & plasmid extraction → PCR identification of positives → Sending for sequencing → Sequencing result analysis.