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) |