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I would like to insert a 6 kbp construct, which I have on a plasmid into the genome of S. cerevisiae. This plasmid was originally constructed to integrate at the HIS locus via homologous recombination after transformation with the plasmid. The problem is that the strain I would like to use is HIS-Δ0, so the complete HIS-ORF has been deleted so that the plasmid will not insert via homologous recombination.

My question is now whether I can still insert my construct into the genome via PCR based homologous recombination, by amplifying the region of interest on the plasmid with primers which have a homology to the genomic sequence where I would like to insert my construct. I am afraid that 6 kbp are too much to transform yeast cells efficiently. Does anyone have experience with such long inserts?

thanks for your help, Jannis

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  • $\begingroup$ Are you having trouble with the amplification or the transformation? $\endgroup$
    – March Ho
    Commented Mar 4, 2015 at 21:04
  • $\begingroup$ No, I have not even tried yet. I am just worried that the homologous recombination will not work with such a long insert and I wanted to know if other people have done similar tranformations before. $\endgroup$
    – Jannis
    Commented Mar 5, 2015 at 10:20

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