Hi, I'm trying to make random mutagenesis on my interest gene. I want to use error-prone PCR. But I only know how to amplify and set condition for best mutagenesis rate. I wonder how can I ligate these products after amplification to my desired plasmid? Can I use infusion or just ligation by T4 ligase?
hi,
you can either do blunt end ligation or sticky end ligation. Obviously for sticky end sequences that are mutated in the restriction sites will not be ligated appropriately
Good luck! Sven
Sven Reister Yeah，thanks for your answer, Sven. If I generate a sticky restriction sites in my gene, I will also failed to ligate that sequence. So I prefer to choose homology methods to ligate PCR products and vector. Gibson assembly or Infusion will be fine.
Hi, you can use both Gibson or traditional restriction based ligation (using sticky end)-whatever you prefer. Do not worry about mutating your restriction site - this is introduced by primers (not amplified during PCR) and wont be mutated.
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