For interfacial studies, I would like to build an alanine scanning library for one of my proteins examining 20 sites. I will ultimately express the gene using E.coli cell-free protein synthesis. I already have the template gene which was originally built using PCR assembly. Now, what's the best way to build a scanning library?

  • $\begingroup$ do you mean, how do i decide which amino acids to include in the alanine scan or how do i create the library of alanine mutants? $\endgroup$
    – shigeta
    May 17 '12 at 19:54
  • $\begingroup$ @shigeta, definitely more "how". $\endgroup$
    – bobthejoe
    May 17 '12 at 21:28
  • 1
    $\begingroup$ genscript.com/alanine_scanning.html genscript has some pages to help you reason through the design. Myself I'm not sure, but it looks like it takes quite a lot of oligos to say the least... $\endgroup$
    – shigeta
    May 18 '12 at 6:08
  • $\begingroup$ Are you still interested in the answer of this question? $\endgroup$ Feb 25 '14 at 20:25
  • $\begingroup$ @GerganaVandova, not really but it might be of interest to the broader community. $\endgroup$
    – bobthejoe
    Mar 3 '14 at 21:08

The fastest will change as time passes and better technologies are developed.

I think the fastest method existing at the moment is Shotgun Mutagenesis (provided by Integral Molecular Inc).

This does not employ any new method of doing that. They just provide a set of plasmids, that has all the possible mutations. The set itself is generated by automated DNA synthesis.

So if you don't have a DNA synthesizer with you then simply order the kit from the company.


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