I used pHEN2 phagemid and CM13d3 hyper phage to construct a phage library with 5 randomized amino acids. When I sequence the library, there is stop codon showed up within the 5 randomized aa area. From my knowledge, the phagemid is expressing the piii protein with the randomized AAs at the N-terminal. If there is a stop codon at AA site, the piii protein should not be express and should not construct to a phage. But I am not only seeing the stop codon but also the chances I saw it is increase after I amplify my phage.
If anyone meet this problem, please let me know the reason and how I can solve this problem.
Best,
Yuhan
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