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[Arabidopsis] question on Y1H

Smith Chiang via arab-gen%40net.bio.net (by chiangscn from gmail.com)
Sun Jul 15 07:19:14 EST 2012


Hello colleagues,

I want to test the binding of a TF to its putative target gene
promoter using Y1H (not EMSA right now).  For the promoter fragment
cloned into pHIS2 or similar vector, is it ok to clone the whole
promoter fragment (i.e. 1 kb) upstream of ATG start codon, instead of
using a tandem repeated, short fragment?  Is this feasible? Does ayone
ever do such work?

If I have to use short, tandem repeated fragment containing the
cis-element, how to construct such a tandem repeated fragment(eg, 4
times repeat) into a vector like pHIS2? synthesize a long primer and
run PCR, cut and then ligate them together? is there any detailed
protocol to do such work?

your reply is greatly appreciated.

thanks and regards,

Smith



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