can I amplify FOXO1 and RUNX2 with proof reading polymerase such as high-quality Taq polymerase. Then, incubate the PCR product with ordinary Taq polymerase to generate A overhang
Also, I would like to use Dual luciferase assay but I am not sure how to construct the vector and add the moG2 promoter??
any idea??
Ghada,
Yes, this is the commonly accepted way. Use a proofreading enzyme (such as Pfu, Q5 or similar not Taq!) to amplify your gene then A-tail with Taq polymerase and dATP.
The quickest way to doing cloning of several pieces together is Seamless cloning (such as Gibson assembly or other proprietary version of it). I'm not clear which Dual luciferase assays you plan to use and exactly how you want the vector to look, so I can't be more specific.
Ghada
Alternatively you could try to directly clone your inserts into a blunt site (SmaI/EcoRV) of the vector. In any case (blunt or A-tailed), remember that only half of the clones will cary the insert in the required orientation so you will have to work it out..
Need to know more details on the dual-luciferase cloning to answer.
Sorry, I didn.t see you are using a topo plasmid. They only accept A-tailed inserts. Forget what I said about blunt cloning.
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