I'm having a problem growing my transformants (from golden gate assembly) in LB broth with 50 ug Kan (recommended concentration using the plasmid backbone). The colonies were from LB plates with 50 ug Kan (in which colonies form 24-48 hrs)
Saw a suggestion somewhere here to check competent cells and the media: seems its not the problem because the competent cell can take in the plasmid vector (control) with Kan gene well and grows well in plates and broth (16-20 hrs is okay)
I tried checking the ligation product from golden gate assembly for the insert using PCR with the plasmid vector as control-- I get my expected size from the golden gate assembly (~2kb insert) and 3kb from the control. I also checked after purifying the control plasmid from the transformation- I still get the 3kb band size.
The problem is my ligation seem okay since I can get the 2kb insert after checking ligation product. Growing in LB plates is slow but I cannot grow at all in LB broth.
Hmm, if the colonies are growing on plates but not in liquid that sounds like a possible incubator problem. Double check the temperature (put in a thermometer, don't just rely on a dial/settings). If it's too hot then your cells will die (mine did that during a summer heat wave).
Maybe try growing a different strain or two at the same time to see if it is a general or a specific problem.
Fresh LB Kan would be a good idea too.
if growing on plates is much slower than the control plasmid grows on plates, then it sounds like your insert might be a bit toxic. This could be why you are not getting good growth in liquid. If there is an IPTG inducible promoter on. your plasmid, then adding glucose to the medium might reduced expression and get you a bit better growth.
You could also patch out your colonies onto quadrants of an LB-kan plate and scrape them up with a sterile toothpick later instead of growing in liquid. You can miniprep from the resuspended colony mass.
Agree with Michael J. Benedik , please also try to do multiple screening to check you isolating healthy colonies and double-check for signs if there is any contamination in your culture flasks. Try doing empty autoclaving of your flasks before preparing media as well.
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