I'm interested in amplifying a sequence for further use with Gibson Assembly. I want to create overhang regions in my DNA fragment so there would be complementarity to the plasmid I'm trying to insert it in.

I was informed by a colleague to work with "flagged primers" though I can't find any literature online. Can anyone explain this concept to me?

  • $\begingroup$ Are you sure they didn't mean labeled primers. The Oligonucleotides become part of your DNA, so if they are labeled, then the label will be incorporated into your amplified sample. $\endgroup$ – AMR Dec 19 '15 at 0:01
  • $\begingroup$ they described them as forward and reverse primers with overhang regions $\endgroup$ – John Dec 19 '15 at 3:32
  • $\begingroup$ The only place I could find a reference to flagged was on what appeared to be an personal journal from someone at UCSC. This is the Addgene protocol. If has a link to the Gibson's original 2009 article, and that doesn't mention flagged either. Nor does Janet B. Matsen:Guide to Gibson Assembly $\endgroup$ – AMR Dec 19 '15 at 6:56
  • $\begingroup$ There is nothing, in my knowledge, called flagged primers. What your colleague must have meant is perhaps "flanks". If you clarify your objective then this question may be answered. Since the real question is unclear as of now, I am putting this on hold. $\endgroup$ – WYSIWYG Feb 11 '16 at 9:17