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In the GeneEditor™ in vitro Site-Directed Mutagenesis System, which one of the
two selection oligonucleotides should I use with my specific mutagenic oligonucleotide
designed to mutate the gene insert?
The GeneEditor™ in vitro Site-Directed Mutagenesis System(a)
(Cat.# Q9280)
requires all oligonucleotides used for mutation to be complementary to the same DNA
strand. The orientation of the cDNA insert and Ampr genes can therefore be used
as references to determine which selection oligonucleotide to use.
- Ampr gene and inserted gene are in the same orientation. If the mutagenic
oligonucleotide A (same 5´--> 3´ orientation as
the insert) is used, the TOP strand selection
oligonucleotide should be used in the mutagenesis reaction. (See the figure below.) If the
mutagenic oligonucleotide B (which has the opposite
orientation to the insert) is used, the BOTTOM strand
selection oligonucleotide should be used.
- Ampr gene and insert are in the opposite orientation. If the mutagenic
oligonucleotide A is used, the BOTTOM
strand selection oligonucleotide should be used in the mutagenesis reaction. If the
mutagenic oligonucleotide B is used, the TOP strand selection oligonucleotide should be used.
(a)U.S. Pat. No. 5,780,270.
GeneEditor is a trademark of Promega Corporation.
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