Hello,
I am concerned about my cloning.
I am trying to clone a 1.7kb fragment into my 3kb backbone. When transformed and plated, the cloning seems to work, but the background colonies are abundant - for all of no ligase, no insert and no backbone controls. Note however, the no backbone controls had 1/2 the background than the no ligase and no insert.
Going backwards, to clone the 1.7kb fragment in, the 3kb backbone had two digest sites next two each other (spanning 12bps), and when double-digested and phosphatased, the double-digested control also had a ton of background.
However, when I performed maxiprep on the outgrowth of the bacteria containing the 1.7kb+3kb clones, and digested with individual enzymes, I saw a clean band at the expected size. Moreover, when I double-digested, I was able to drop the 1.7kb fragment back out.
So back to the question, if those bands from maxiprep on a 1% agarose gel are clean, why do my plates have so much background?
Yes, I have also considered the antibiotic not working, however it is as I plated the same bacteria on the same batch of plates and nothing grew.
Thanks in advance!
Stan
Hi Stan,
what type of antibiotic do you use? If it is amp you might have trouble with satellite clones. Here you can find more information: http://bitesizebio.com/10188/whats-the-problem-with-ampicillin-selection/
Best, Christian
Hello Christian,
I use carbenicillin as my antibiotic as I'm aware of the ampicillin-satellite colonies issue.
Best,
Stan
If you get growth in a non-plasmid-at-all control, you have contamination, obviously. Smell that control dish; does it seem to be coli or yeast? Have you tried to grow one of such "control" colonies?
From where are you getting the 1.7 kb insert? Are you digesting it out and gel-purifying from another vector? In that case, is there a chance that the recombinant colonies you are getting in the backbone+insert reaction are actually carryover plasmids from the previous construct?
@Ricardo
I am miniprepping the background clones to find out exactly what size it corresponds to, to try and find out what it is.
@Alejandro, the insert is indeed from a donor plasmid. I initially thought the background on the no backbone may be from unclean gel extraction of the 1.7kb fragment, so I redid it. Then I transformed the old insert I used by itself and the new insert that I redid (gel extracted), and saw that the old insert indeed yielded more colonies, but not substantially more (i.e about 13-15 versus 2-3 on the new insert plate).
I am almost certain it's coming from the backbone, which is already a plasmid library that has been cloned into a backbone plasmid purchased off Addgene.
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