Template DNA mutates.(SNP, insertion, deletion)


 

Status descriptions

  • It appears double signals for a single base.
  • All wave peaks are clear, but there are two overlapping wave peaks at certain sites.

Possible reasons

  • The PCR DNA mutates.
  • When the plasmid construct is poisonous to the bacteria, the DNA often mutates in the process of cloning.

Solutions

  • 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.