Table 8.

Troubleshooting.

Problems	Solution
Step 7, No colony after transformation	The ratio of insert to vector was not adequate. In step 3, the ratio of insert to vector is 40:1. It can be varied from 10-100:1.
Step 8, No visible difference of bands	Always use at least 3% agarose gel and run for 2-3 hrs at 180 V.
Step 9, Sequencing does not work	Sometimes, it is difficult to read through the hairpin because of its strong secondary structure. We have tried a few companies and found Genewiz Inc. can read through the hairpin structure with good results.
Step 10, No decreased expression of the target gene	It can be caused by poor transfection efficiency. Try to find an adequate protocol for transfection. Another possibility is that the RNAi oligo does not work. If this is the case, new RNAi oligo should be used. We normally start with oligos against two different regions of the target gene and rarely found both do not work.