This protocol resulted in extremely high transformation yields (approximately 10 individual clones per transformation).
The recovery coefficient was also higher for higher background activities.
Katahdin showed the highest transformation efficiency.
When we employed conventional transformation using only one vector, we obtained much higher transformation efficiency.
These reports showed a very high background transformation rate in the NIH 3T3 cells (50 foci per μg control DNA, ref. [ 24]; see photographs of cell culture dishes in Fig. 2 of ref. [ 23]) casting doubt on the reliability of the transformation assay.
But, attempts to knock-in genes in some cases have encountered a high background of transformation due to maintenance of unwanted circularization of the transforming DNA, which contains possible autonomously replicating sequence (ARS) activity.
However, several attempts to knock-in an Mmc gene into the Mmc genome were inefficient with a high background of transformation.
Unless a very flat subclone of NIH 3T3 cells is used in these assays, as we used here, a high and variable background transformation rate can be observed.
Although TREC method is currently the best tool that can seamlessly engineer a genome cloned in yeast [ 21, 22], the process is sometimes inefficient with a high background of transformation, arising possibly due to illegitimate recombination.
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