We are having trouble transforming plasmids into clinical strains of E. coli by electroporation. We use a 1500 V pulse and standard electroporation buffer and follow the protocol that comes with our Eppendorf Gene Pulser. Are there any known pitfalls to this process? I should perhaps mention that these are mecillinam resistant clinical isolates; they are slow growing and we suspect that they are defective in cell elongation.
Some few questions: Did you carry out a control electrotransformation,when you are working with clinical strains is vital you set up a control transformation with laboratory strain-that is restriction deficient, what is the copy number of your plasmid? How long did you carry out recovery? What is the antibiotic cassette on the plasmid , since you are working with E.coli I assumed you are using ampicillin selection, in that case you could start selection with a reported concentration for selection with this antibiotic . Transformation has so many pitfalls unless details I provided we could only speculate.
I would suggest 2500V pulse with 2mM cuvette and 30 microlitre cells. give 45 mins growth and plate in media with suitable antibiotics
I agree with Priyabrata. I also observed increased amount of trasformants for clinical E.coli with voltage increase. I would also extend the growth time after electroporation before plating bacteria on selective agar to 6-12h, especially that you mention that they are slow growing bugs. Also I would incubate plates for 48h and then check for transformants.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology