I transformed DH5alpha bacteria 2 months ago. I got some colonies on kanamycin plate. I picked a colony and used plasmid after miniprep. Plasmid worked fine but I forgot to make glycerol stock. Now I am trying to grow bacteria again from same plate which was kept at 4C all the time. They are not growing at all. What could be the reason?
Ehm.. 4ºC.. are you sure about that? Compared to environmental strains, E. coli is unfortunately a bit fragile concerning durability. Cold rooms generally labeled as being at 4ºC are sometimes in fact at 5-6ºC, so the metabolism is not quite dormant. If you're talking of a regular freezer, then it's worse: around 10ºC. Therefore: you should never store an E. coli which you intend to work with again for more than 1 month. 2 months, in my experience is stretching their survival next to the limit. You should have streaked part of the material of that original colony to a new plate with the desired antibiotic (i usually use for example a sterile toothpick or a micropipette tip), and then inoculate the tube for the mini-prep. That way you always insure a backup with enough material (i usually make a grid on my backup plate, so it can fit around 50 clones).
Now.. solutions to your problem. I hope you still have the tube with the plasmid you obtained after that mini-prep. Transform a competent E. coli ASAP with that plasmid. And then, don't forget to make the stock ;).
If you don't, then you're in trouble. Good luck!
Ehm.. 4ºC.. are you sure about that? Compared to environmental strains, E. coli is unfortunately a bit fragile concerning durability. Cold rooms generally labeled as being at 4ºC are sometimes in fact at 5-6ºC, so the metabolism is not quite dormant. If you're talking of a regular freezer, then it's worse: around 10ºC. Therefore: you should never store an E. coli which you intend to work with again for more than 1 month. 2 months, in my experience is stretching their survival next to the limit. You should have streaked part of the material of that original colony to a new plate with the desired antibiotic (i usually use for example a sterile toothpick or a micropipette tip), and then inoculate the tube for the mini-prep. That way you always insure a backup with enough material (i usually make a grid on my backup plate, so it can fit around 50 clones).
Now.. solutions to your problem. I hope you still have the tube with the plasmid you obtained after that mini-prep. Transform a competent E. coli ASAP with that plasmid. And then, don't forget to make the stock ;).
If you don't, then you're in trouble. Good luck!
2 months is too long for +4C. if you storage the bacteria you should make glycerole stocks and then store at -20 or -70. it provides long storage then plate.
Maybe you tried to culture false colony (not resistance antibiotic; in plates some false colonies may be observed which doesnt obtain your antibiotic resistant plasmid))
DH5alpha like most molecular biology strains have a mutation rendering them RecA-. Such strains die quite rapidly and have a lifespan of only a few weeks on a plate at 4C. You probably should just retransform from your miniprep DNA. If you no longer have your plasmid prep, you could scrape some dead cell mass from the plate, boil it to lyse the cells and retransform with a small aliquot
try to take for growing big number of colonies not one or two and give them more time of incubation.In most cases it works
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