2×10^5 recombinants/10^8 viable cells
||Bacteria Escherichia coli
||Ellis HM, Yu D, DiTizio T, Court DL. High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci U S A. 2001 Jun 5 98(12):6742-6PubMed ID11381128
||Researchers wished to see whether the ? Red system could use a 70-base single-stranded oligo to correct the amber mutation galKtyr145am located on the bacterial chromosome. For this purpose, they synthesized an oligo corresponding to the non-template DNA strand of galK. Strains HME6 and HME9 carrying the amber mutation were induced for ? Red expression by shifting the culture to 42°C for 15 min and were prepared for electroporation as described by Yu et al. 2000, PMID 10811905. These cells were then electroporated with the “wild-type” oligo and plated on minimal galactose plates to select for Gal+ recombinants. They obtained ˜2×10^5 Gal+ recombinants per 10^8 viable cells by using 10 ng of oligo.
||To determine the minimum length of homology for recombination with a single-stranded oligo, researchers compared the ability of oligos from 20 to 70 bases long to generate Gal+ recombinants in the galKtyr145am strain HME9. The 60-, 50-, and 40-base oligos recombined less efficiently, yielding 4–5×10^4 recombinants per 10^8 cells. Recombination efficiency dropped to 5×10^3 recombinants per 10^8 viable cells with the 30-base oligo, and Gal+ colonies were near background levels with a 20-base oligo.