Multiple in vitro mutagenesis reactions

Rich Dudley rdudley+ at pitt.edu
Wed Nov 17 08:37:24 EST 1999


We did 9/9 in one reaction with Promega's pAlter kit, and most of the
colonies had 9/9.  It did involve making ssDNA and switching antibiotic
resistances from Amp to Tet (this was 2/9 of the mutations--you could
probably use their GeneEditor primers to cut down on the work a bit).  This
method is a little tedious, but I prefer it for multiple, simultaneous
mutations.  Depending on how far apart your mutations are, you might have to
do several rounds to avoid gigantic oligonucleotides.  We didn't use uracil,
just the antibiotic selection.  If you can afford it, have your primers
synthesized with 5' phosphates--we found that to be many orders of magnitude
more efficient.

Good luck!

rich






More information about the Methods mailing list