I use the psip-403 vector that contains the erythromycin resistance gene for cloning, and I also tested several strains of Escherichia coli bacteria (top10f, dh5 alpha) for this purpose, and the cloning results were that both cloning DH5-a harboring erythromycin resistance gene and non-cloning DH5-a survive in erythromycin contained agar plate. Im sure that the cloning was successful and I tested and cultured different concentrations of the antibiotic erythromycin in the agar medium (1-0.2 g of antibiotic in 1 ml of ethanol for 50 ml culture medium). Also, I tested two different brands of antibiotics (molecule, sigma and pharmacy antibiotic tablet form) and used different solvents such as ethanol and dmso, but the results were still the same. If possible, please guide me in this matter.
Hello, and I hope everything goes well.
Your issue may be because of several things:
- It's crucial to check your antibiotic for expiration or degradation (in our institute, most antibiotics were bought 20 years ago), especially in case of heat.
- Check the concentration of your Antibiotic (100-250 ug/l is appropriate)
- At a proper temperature, antibiotics must be added to the media. (not so hot temperature that antibiotic degraded and not so cold that is not getting spread).
- Be sure your pure E. Coli strain doesn't have a resistance gene & and has not been mutated (you can check it by sequencing methods
- Your DH5α may have accidentally received a plasmid containing a resistance gene to erythromycin...
- another common mistake is to forget to add antibiotics.
It is probably caused by your antibiotic and the solution. To check your antibiotic acts correctly, make five plates as below and check them all:
Ignore the answer from Mahdi, it's AI-generated nonsense.
Your antibiotic selection strength is not correct and you are using WAY too much solvent for the volume of media.
Here's a link to a lab website with instructors for how to make many of the common selection media types.
https://barricklab.org/twiki/bin/view/Lab/ProtocolsAntibioticStockSolutions
Katie A S Burnette
Hello Katie,
To clarify your misunderstanding about considering me as a bot, all the steps I described were my experiences in the lab based on trial and error. However, I took answers from them.
Here is my LinkedIn profile for any more discussion :
www.linkedin.com/in/mziarazzaz
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