I cloned a gene into a vector with Kanamycin resistance and plate it on a Kanamycin plate and get many colonies. I then did colonyPCR followed by miniprep (I kept glycerol stock for those colonies that I did miniprep for) sent them for sequencing and got the correct sequence.
Then, when I tried to do a midiprep (200mL) using the glycerol stock of the colony with the correct sequence, nothing grows in my LB Media eventhough I used the same batch of Kanamycin (50mg/mL stock, so I add 200uL into 200mL of LB). I've tried this 3 times and everytime, nothing grows. I scraped a bit of the glycerol stock using a pipette tip and threw the whole tip into the media and grew at 37 degree overnight, shaking at 200rpm.
The first time didn't work so I tried again, this time using half the concentration of Kanamycin. Still, nothing grew. The 3rd time, instead of scraping from the glycerol stock (thinking that I might have only scraped teh glycerol instead of the cells), I thawed the glycerol stock and took 10uL of the thawed cells and threw the whole tip into the media, but still, the next day, the LB was clear.
Have someone had the same experience? How did you solve it? Is there any suggestions that I can try?
Thank you very much in advance.
Hello William,
Apparently a quite late answer, so I hope you could solve it by yourself, but just in case...
The only time I've seen that behaviour, in otherwise controlled conditions, was when a student was growing a medium sized culture (50ml) in several falcon tubes, and had screwed the caps tightly. No air, no growth. That kind of obvious/unsuspected details. So for that part, always put at most 20% of the flask volume worth of medium to ensure aeration, and make sure air can come in the flask.
it could also be, as you suspected, that the glycerol stock isn't good anymore/had a problem in the making process. The best way to test would be to try a fresh transformation with the plasmid you miniprepped at the beginning, after the PCR (you only need 1ul of miniprep for that, usually, so you should be fine). Another test for that would be to inoculate the glycerol stock in LB without antibiotics, with a side control where you fake-inoculate LB, without bacteria, to see whether the bacteria in the glycerol stock survived or not.
Assuming a "harmless" plasmid (not causing the cell to express something toxic at a basal level), that's so far all I could come up with.
Good luck!
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