Hello, I need help improving my counterselection procedure when using sac-B. I have grown my E coli strain of interest in LB-agar media with and without sucrose (5%), at 30°C, following the procedure described by Hagg et al (2004). Surprisingly, I find more colonies in the plates containing sucrose than in the ones without sucrose, which is very intriguing. I now find many mentions of sucrose working better in media without NaCl. Does anyone reccomend some other modification to my protocol? Should I use a higher sucrose concentration in my plates?
Thanks in advance for you help!
Celina
I have always used 10% sucrose and LB with low NaCl (you can use 1g NaCl instead of 10g per liter to make your LB).
Many thanks Michael J. Benedik for your answer. I will be trying the media composition you reccomend, best regards!
You may also find that it works better in some strains than in others. So if the new media doesn't help, try a different host strain if you can.