I frequently need to do plasmid preparation and maxiprep does not work so we stick with miniprep. For our bacteria stock, we kept it in glycerol and usually inoculate them straight from the glycerol and grow them in LB broth overnight. (We does this around once per 2 weeks). It worked for 2-3 times and after that, even stock that were not touched cannot work anymore. I tried streaking them on plate from the glycerol stock and no colony growth was observed. Any idea on how to troubleshoot? Thank you.
I suggest to change the culture medium and using Circlegrow medium is recommended.
How was stock made? There are following two type you can make, which will allow survival of bacterial strain for 6-12 month.
1. From Plate: Streak E. coli on LB to get mat growth, scrap bacterial colony mass and put in to vial containing 1 mL 20% sterile glycerol (make 2 stock from one plate). Slightly vortex to mix and put at -20 C temp (deep freeze)
2. From Broth: Take 0.5 mL active log phage culture of E. coli grown on LB and put in to vial containing 50% sterile glycerol.Slightly vortex to mix and put at -20 C temp (deep freeze)
Note: Glycerol shroud be autoclave twice for 30 min at 121 C temp.
Incubate fresh competent cells with your plasmid (~20ng) for 1hrs, spread on plate to grow colonies overnight, thereafter inoculate for plasmid preparation or use these cells directly for inoculation for plasmid prep. Hope this will be easy solution for your problem.
Thanks
Glycerol should autoclave because spores (Bacillus etc) are more stable to glycerol.
You can stock several aliquiots and use one tube only for one time.....
I suggest to streak first on LB plate and allow it to grow for 2-3 days, if grow, you can make culture in LB broth.
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