- It appears double signals for a single base.
- All wave peaks are clear, but there are two overlapping wave peaks at certain sites.
- The PCR DNA mutates.
- When the plasmid construct is poisonous to the bacteria, the DNA often mutates in the process of cloning.
- If the template DNA is heterozygous, connect the PCR product to the vector first and then make sequencing.
- If the plasmid construct is poisonous to the bacteria, replace with a vector in low copy number or replace the bacteria (cell) with another strain.
- Use the primer at the other end to make sequencing.