FAQ 3: What happens if I grow the recombineering cells at temperatures higher than 32°C?

ANSWER 3: If you make the mistake and grow your overnight at too high of a temperature, you should abandon the experiment and start over. Growing the cells at >34° will induce the pL operon which does several things. If you are using a λ prophage containing strain, the kil gene is expressed, which will kill the cells if made for an hour or more. Extended expression of Gam will also kill cells. Thus, there is a strong selection for mutations that prevent expression of pL when cells are grown at elevated temperatures.  Once the pL operon is inactivated, the cells will survive but will be useless for recombination, since Red will not be expressed! There are exceptions: for example, strain SIMD50 does not contain either the gam or kil gene and thus there is no problem. Also, the pSIM plasmids do not contain kil. However, many of the pSIM plasmids have temperature-sensitive replication origins so you may lose your recombineering plasmid if the strain is grown at too high a temperature. The bottom line is, know your cells genotype and what it means! It is always safer to routinely (as we do) grow your recombineering cells at 32°.