Hello,
I picked colonies and inoculated in 5 ml of freshly made LB medium + Ampicillin for Miniprep before Maxi. About 17 hours incubation. There is a very weird string like structure in my culture flask. Anyone experienced this before? The other colonies do have some debris in them, seems shorter string, but not as long as this thing. Hopefully you can see from picture. Took with my iphone with macro lens.
Worth keeping it ? We need the bacteria for the plasmid. Tried many times to get them, at least this grow very well. Really wanted to use it for Maxi.
Thanks!
2 possibilities -a) contamination, b) an antibiotic effect,- probably b) Remove a drop and look under a microscope. You may see long almost "filamentous" bacteria rather than the normal (E.coli ? ) bacteria shape. I think that under- expression of the antibiotic resistance gene leads to interference with normal antibiotic-resistant growth. You may want to re-streak your colonies on freshly made antibiotic plates and start again. Use freshly made antibiotics. Are you using a low copy vector?
My guess is that the cultures started off looking OK, though growth was a bit slow (whispy). As it progressed the strings started appearing and in some cases it looked like the bacteria had almost lysed giving rise to a slightly "gloopy" mess and a nearly clear suspension. You may be able to salvage some plasmid from the material if you can pellet it effectively, but it may not be the best material to proceed with.
Hello,
Just an update to the situation. I had a look under the microscope. The organism present looks like a rod shaped structure, I found a picture by googling that best represents it. I would say 40% of them are moving like worm crawling, others are stationary.
I plate some onto an +Amp plate and they grow as well. and I just scrap the bacteria from plate and along with the bacteria from the tube, I did mini preps.
None of them were not carrying my plasmid. Nothing obtained from DNA staining, just smear. Not sure how they manage to grow though.
But thanks a lot for all your advice and helps !!
I presume you mean none were carrying plasmid. Your amp is probably past its best or when you made the plates it was added into the agar when too hot. A few bacteria managed to cling on and form "colonies" but without plasmid present when they were transferred to culture were unable to grow properly - some lysed - others failed to divide properly. Antibiotic was not sufficiently "strong" to kill them off properly.
Bad news but you may have to start over again.
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