We have recently purchased competent shuffle E. coli cells from NEB. The problem is, they do not want to grow - colonies are really small and when transferred into liquid LB they almost stop growing. We culture them in LB media with 100 µg/ml Ampicillin at 30 °C.
Any suggestions?
Hi Sabina,
You have to be really patient with these cells-the changes to these cells that make them better at re-folding proteins also make them grow very slowly so colonies on plates even at 37°C will only be obvious after 24 hours.
For expression cultures etc. you also have to extend the growth times dramatically the sort of times we have been using are 24-36h for starter cultures (37°C), 6-8h for growth phase after dilution (37°C) and up to 48h or more after induction (e.g. 20°C).
This does make them a bit of a pain to work with especially if you want to compare different bacterial strains with very different growth characteristics but they can make a real difference to some targets
If your expressed proteins are toxic you should also include 1% glucose in all plates and media if you are using T7/IPTG, something we include routinely, to prevent leaky expression prior to induction.
Thank you very much for your response! We were worried that they do not want to grow. I will try to let them grow for longer.
What about antibiotic concentration in the media after such a long time of incubation?
If you are worried about maintaining your antibiotic concentrations I would recommend the use of Carbenicillin in place of Ampicillin, it is far more stable, although you are starting with a relatively high concentration. If you 'shop around' you can also get it for a reasonable price.
I'm using Shuffle and for me it grows well when temperature is 25oC or lower. Also, due its oxidant cytoplasm is better to grow it with low agitation. In plates, the colonies are smaller than E. coli BL21 and you have to wait longer to see colonies. Hope it helps
Hi Sabina, I think you got ur desired result now :D but by the way, for SHuflle 30'C is much better than 37. about Antibiotic, I use spectinomycin, don't have any idea about Amp resistance (if your vector has resistance to Ampicillin, should add Amp and Spect to your media). LB is suitable media but as Dr. Nick S Berrow mentioned it has a slow growth rate. you will get very small colonies on solid media and takes a longer time to grow in comparison to other E.coli bacteria strains.
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