How to analyze the sequencing results after PCR? Please be more detailed, thank you!

First, look at the sequencing peak diagram. If the color diagram of the result shows that the peak shape is sharp and single, and the codes corresponding to the bases are consistent, then it can be judged that the sequence can be used. If there are abnormal phenomena such as double peaks or signal interruption, it needs to be prepared again or cloned before sequencing.

Secondly, blast the PCR product on NCBI to see if it matches the fragment you expect. If the match is consistent, you can proceed to the next step, such as cloning, expression, etc. If the match is not high, it is recommended to prepare it again. It is best to use methods such as touchdown or nested PCR to improve the specificity of the product and then amplify and sequence it.

In addition, if you are doing SNP analysis, you need to look at the sequencing color chart to find the corresponding mutation heterozygous site.