Table 7.

Troubleshooting Guide for DNA Blotting and Hybridization Analysis

Problem	Possible Cause	Solution
Failure to generate lentivirus or very low virus titers	1. 293 cells at high passage numbers, or in suboptimal growth condition 2. Transfection failure due to temperature and pH variability.	1. Use lower-passage number 293 cells (<20). Cells should be between 50~80% confluent at transfection. 2. CaCl2 and HBS should be kept at 4°C and warmed up before use. pH the HBS periodically to 7.1. 3. Use plasmid expressing fluorescent protein as positive control. 293 cells (and phenol red-free medium) should turn to corresponding color if transfection succeed.
No knockout or partial knockout	1. If using antibody-based validation methods (western-blot, immunofluorescence), note that antibody may be non-specific to the target protein. 2. sgRNA not active or does not result in complete gene disruption.	1. Use different antibodies, especially ones that have been knockout or knockdown validated. Otherwise, use a DNA-based validation approach. 2. Try different sgRNAs. Use multiplex sgRNAs cloned to a single plasmid to enable large fragment deletion (Cong et al., 2013). However, if the target gene is lethal to cell survival, then complete knockout of that gene may not be possible.