Dear Friends,
I am planning to do yeast one hybrid experiment to analyze one of our Oryza Sativa (rice) promoter sequence through Yeast one Hybrid kit supplied by clonetech.
The difficulty is that as per manual we should not go to take more than 100 bp fragment for analysis in our pAbAi vector as it may gives false positive screening in Aeurobasidin plate.
I don't have any idea regarding my 1000 bp promoter sequence that which one is active element regarding the gene expression in my case. The overall fragment we have planned to clone it in 100-200bp size length in overlapping manner from staring +1 to +1000 bp for full length promoter analysis.
Has anyone tried with such a big fragment like 100 to 200 bp sequence in bait vector without facing very much difficulty for false positive screening before cDNA library transformation.
Please give your views if you have any idea.
The kit manual file I have attached with this description.
It depends upon each individual promoter sequence. My suggestions as per experience is to first check;
1. Clone your ~1Kb promoter region, except 50-70 bp basal promoter region around transcription start site, in pAbai vector.
2. Integrate this construct in Y1H Gold strain.
2. Check the reporter gene activity and confirm upto what Aureobasidin A concentration you can stop yeast growth in presence of control pGADT7 vector only.
3. Screen your cDNA library at this Aureobasidin A concentration.
Good luck
Thank you kamal sir for your nice suggestions.
Definitely I will start my work as you suggested.
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