I'm currently using Golden Gate cloning to assemble transcription units (promoter, CDS, terminator) following Deuber et al(2015). Problem1: Lot of false positive white colonies, that actually have RFP containing vector but is still white
Problem 2: Even when I tried breaking the cloning into 2 steps (insert the GFP transcription unit and then insert my own promoter, CDS, terminator), the green colonies, which apparently have the GFP and other parts inserted are not the correct constructs. the connector and other sequences are absent form those green colony plasmids.
Please help.
Hi, I do not know the yeast system, but do use MoClo all the time. If you do have false positives on your plate as your selection marker RFP does not seem to be perfectly clear, you can make a terminal digestion after the golden gate reaction using the same restriction enzyme.
Otherwise, I do not understand what you mean with breaking into two steps. Golden gate cloning works so efficiently that you should not have any trouble assembling three parts. I would therefore guess that you do have a conceptual mistake - wrong vector, enzyme, overhangs or design. If you do the right thing 99% of all clones will be correct.
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