Table 2.

Troubleshooting table

Steps	Problem	Possible reason	Solution
11	Small size RCA products	DNA cut from the gel is not circular supercoiled	Repeat Step 7; cut the band with the fastest mobility from the gel again
30	Poor transformation efficiency	Spheroplasts are not competent for transformation	Make spheroplasts according to the protocol
30	No yeast transformants	Yeast cells were plated onto wrong medium	Ensure that the medium contains all required nutrients
30	Poor transformation efficiency	TAR vector was phenol/chloroform purified	Vector should be column-purified
30	Poor recombinational cloning	Amount of the TAR vector is more than 40 ng	Check concentration of the vector
30	Poor recombinational cloning	Amount of RCA products is less than 2 μg	Check concentration of the RCA products
30	Poor recombinational cloning	Sequence homology between hooks and the repeats is less than 90%	Change the hooks in a TAR vector
67	No arrays in the vector	Wrong orientation of the hooks in the TAR vector	Orientation of the hooks should correspond to that illustrated in Figure 1c
67	Unstable arrays	Growth of E. coli transformants at 37°C	Grow the cells at 30°C
67	Unstable arrays	Growth of E. coli culture with good aeration	Grow the cells with a slow shaking
89	No increase in array size after additional recombinational cloning	Cut sites of endonucleases are too far from the ends of the array	Choose endonucleases that cut closer to the end of the array (< 20 bp)