I'm looking to do a luciferase reporter assay with a mutated and unmutated version of the binding sites for my GOI. I want to get a multimeric binding site cloned in, preferably 4-5 repeats of an ~30 BP region. I can't find anything that doesn't charge an arm and a leg for synthesis of something like that, but maybe I'm just looking in the wrong place. Alternately, are there other good methods to make this on my own? I need to make sure I have the same number of repeats in both of my constructs, which makes it difficult to do on my own. Thanks!
Dear Ethan,
the simplest and even cheapest and least time consuming way could be de-novo Synthesis by a company. Otherwise you clone it with blunt Ends and have much of screening to do. The Problem is not to have comparable amounts of inserts, but you need very much luck that all are in the same direction. With three repeats it could be possible.
@Mario Krehan, do you have a recommendation for what company/service offers this for a decent price? Everything I've found has been unfeasibly expensive.
Dear Ethan,
try MWG Eurofins. It could be more expensive if you introduce such repetitive sequences. (I am not entirely sure, I think it was ~300€ up to 900bp.)
how did you solve the problem?
I am just starting the Luciferase assay. I don't know how to start. I have only GOI and its downstream gene. how can I find the binding site and design for the assay?
Anyone, please feel free to teach me.
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