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FAQ 14: Why don’t I get the high frequencies that you report for ssDNA oligo recombineering?


Answer 14: A number of factors can account for problems in obtaining high oligo recombination frequencies, most of which are described in Sawitzke et al., 2011. We find that the final recombination efficiency you get results from the cumulative effect of these different factors, with each making an independent contribution. If one or more of these parameters are not optimal the recombination frequency can be dramatically reduced.  These parameters include:

1. Problems with DNA uptake.

2. Using an inappropriate oligo length - we use 70 base oligos or longer.

3. Attempting reactions other than correcting point mutations or changes of a few bases (i.e. removing a large piece of DNA is not a high efficiency reaction).

4. Use of a leading-strand oligo rather than a lagging-strand.

5. Use of too little oligonucleotide, which will be subject to degradation by ss-exonucleases.

6. Methyl-directed mismatch repair correction of heteroduplex recombination intermediates.

7. Long outgrowth following electroporation, which reduces the apparent frequency of recombination by about 6- to 10-fold, because it allows complete segregation of the recombinant bacterial chromosome away from its non-recombinant siblings. Also, if the recombinant confers a slow growth phenotype, the mutant will be diluted further.

8. Problems with expression of Red Beta. While convenient, the plasmids described in Datsenko and Wanner (2000) give ~10-fold fewer recombinants than does the λ prophage. A comparison of the two expression systems is given in Datta et al., 2006.

9. Use of a recombinase other than Red Beta. Datta et al. (2008) observed ~100-fold fewer recombinants for RecT-mediated oligo recombination than for that mediated by Beta.