Hello,
I did a transformation a few weeks ago and while only a few bacteria grew, I was able to pick single colonies for a liquid culture. However, I had to repeat this experiment because something didn't work after seeing the sequencing results.
I re-annealed the oligos and repeated the ligation; however, this time the bacteria did not grow at all after the transformation. I decided to leave the plates a few more hours on the incubator to see what would happen. The bacteria grew as shown in the first image. I thought it could be the plates, so I repeated the experiment on a new set of agar plates. This time, no bacteria grew either (2nd image with empty plate). I find it weird some bacteria grows but only after 8 more hours in the incubator.
Liquid culture medium: Terrific broth
Plates: LB agar + Amp
Incubator: 37C
Note: It can't be the digested plasmid because I used it with another oligo duplex and worked well.
Yes, Always do fresh transformation and do not incubate for longer times. They may be false positive so grown on amp- plates.
Amy Johnson
Hi, thank you. I repeated the experiment twice: the first time I repeated the ligation. The second time I repeated the annealing of the oligos. The last time, I used a new set of agar plates. Someone else did a transformation on the same plates days before and it worked fine, so unfortunately it is not the freshness of the plates or antibiotics on the plate as I thought.
Ana Morales Benitez Stbl3 is an E. coli strain but is generally used to reduce recombination between repeated motifs. If you are confident that your competent cells are good and your protocol have worked well before (recently) then most likely the problem is with the oligos, you might confirm your design and order new ones.
What you are seeing in the top figure is most likely a single transformant that was growing on a very wet plate and the moisture spread it out over a wider area than a single colony.
Michael J. Benedik Amy Johnson
Thank you. It seems the oligos could have been the problem. We have solved the issue now. Your explanation about the plates was helpful. I definitely remember them not being fully dried. Again, thank you for the help
You are most welcome Ana Morales Benitez , glad that your problem was solved.
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