Table 2.
Troubleshooting table
| Step | Problem | Possible reason | Solution |
|---|---|---|---|
| ν36–38 | Failed PCR while trying to confirm BAC identity or homology arm amplification | PCR conditions are not optimal | Use FailSafe PCR system that includes 12 different buffer choices. If PCR still does not work, change primers. If it still does not work, choose a different BAC |
| 58–60 | Failed amplification of pLD53.SC2/A-box with R6Kγ ori and A-box 3′ primers in PIR1 bacterial colonies | The ligation of the A-homology arm with the pLD53.SC2 did not work | Repeat homology arm ligation and transformation from Step 49 to 60. If it still does not work use PIR2 chemically competent E. coli |
| 96–98 | Failed PCR amplification of DNA from chlor/amp-resistant colony (BAC modification did not work) | Your pSC53.SC2/A-box plasmid preparation could be contaminated with unknown amp-resistant plasmids Your DH10B BAC competent cells transformed with pSV1.RecA could be contaminated with unknown amp-resistant plasmids |
Transform DH5α competent cells with pSC53.SC2/A-box. If your DNA is clean, nothing should grow on a chlor/amp plate. If colonies appear, remake the pSC53.SC2/A-box DNA from Steps 61 to 73 Electroporate the cells with dH2O instead of vector. If you still get colonies on a chlor/amp plate make the competent cells from Steps 76–90 |
| 119 | BAC DNA yield is low as determined by pulsed-field gel | Volume of bacteria used to inoculate LB broth at Step 94 was not optimal Shaking is too strong during DNA preparation |
Change the inoculation volume of bacteria at Step 101 Be very gentle during DNA preparation |
| 123–125 | BAC DNA is viscous after linearization | DNA concentration is too high | Dilute DNA with injection buffer to a concentration that allows ease of flow (no lower than 0.125 ng μl−1). If DNA remains too sticky or particulate at the most dilute concentration, prepare again from Steps 120 to 125. In our experience, the ease with which DNA can be injected is linked to the quality of the DNA. Taking time to produce clean and non-sticky DNA assures that the highest number of transgenic mice will be produced |
| 126 | No transgenic mice are produced after zygote injection with modified BAC | BAC DNA is of poor quality Problem with injection buffer A lethal passenger gene may be present in the BAC (rare) |
Prepare DNA again from Steps 100 to 125 Prepare the injection buffer again. Do not deviate from the recipe Repeat with a different BAC that does not hold the lethal gene |