do 5′-phosphorylated primers expire?

Viewing 1 reply thread
  • Author
    Posts
    • #13253
      vastgenome
      Participant

      Hi guys,

      A naive question, as i m trying to introduce specific point mutations in the middle of a gene linked in a plasmid. The way i do it is through PCR off the entire plasmid and then reclose the circle. I ran into some problems ligating the linear PCR product, and i was wondering if the modified primers (5′-phosphorylated) could lose the 5′ phosphate through frequent thawing/freezing cycles.

      Thanks!

    • #99545
      jonmoulton
      Participant

      Freeze-thaw won’t break off a covalent bond on a monophosphate, but enzymatic activity could.

Viewing 1 reply thread
  • You must be logged in to reply to this topic.