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. 2011 Oct 7;13:8. doi: 10.1186/1480-9222-13-8

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)