Hi all,
With the strategy of 2-step homologous recombination, I have been trying
to replace the integrated URA3 with a PCR product and then undergo 5-FOA
selection. However, I did not get any desired integrated clone yet.
Instead, I even got stable 5-FOA resistant clones in the control that
has been treated everything the same, including ssDNA, LiOAC and heat
shock but without DNA. Here are my questions:
1.What is the frequency of integration that people usually get?
2. After transformation, should I put the yeast in YPD, Uracil drop out,
or water before plating them on 5-FOA plates, and how long is needed?
Any comment or detail protocol is appreciated.
Thanks,
JQ
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