Hi everyone,
I am currently working on expressing a His-tagged protein of interest using a pHERD20T vector. I have successfully cloned my gene into the vector and am now attempting to electroporate Pseudomonas aeruginosa PAO1K with this construct. However, I am encountering difficulties in obtaining single colonies.
Here are the details of my experiment:
- Plasmid concentrations tested: 50 ng and 100 ng.
- Volumes spread on LB agar + carbenicillin (200 µg/mL) plates: 10 µL, 50 µL, 100 µL, and Rest in addition to streaking out 10 µL.
- Observation: All plates, including those with 10 µL, are showing extensive growth.
- Negative control: Plates with sterile Milli-Q water (no plasmid) show no growth, ruling out contamination.
I prepared the antibiotic carbenicillin(200mg/ml) by dissolving the 2g of carbenicillin powder in sterile Milli-Q water, vortexing to dissolve completely and then adding milliQ to achieve a final volume of 10ml. This was followed by filter sterilizing the solution using a 0.22µm filter, aliquoting it and storing at -20°C. Therefore, I don't think I made any mistakes in preparing the antibiotic.
Despite trying different plasmid concentrations and volumes, I am unable to isolate single colonies. A colleague successfully performed electroporation with 100 ng of this plasmid in the past, so I am unsure if my plasmid concentration is too high or if there is another issue at play.
Has anyone experienced similar problems with this plasmid or have suggestions on how to improve transformation efficiency? Any insights or recommendations would be greatly appreciated.
Thank you!
If your negative control was clean and your transformation has a lawn then maybe it worked too well. Just to confirm, your negative control was an actual electroporation but without any added plasmid DNA?
Why not just take a loopful of cells from any one of the transformation plates and streak it out for single colonies on a fresh plate and not worry about it? Or if you really want a bunch of single colonies then repeat and prepare 10-fold and 100-fold dilutions of your transformation mix before plating.
Hi Michael J. Benedik
Yes, my negative control was an actual electroporation using the same PAO1 culture. Instead of plasmid DNA, I added sterile MilliQ water but performed all the steps of electroporation alongside my other samples containing plasmids.
I also made a dilution series and plated some of the transformation mix. The very few single colonies I obtained were then checked with colony PCR to confirm the presence of my insert, and all the samples showed the band at the correct length. So, you're right—the electroporation was very successful.
However, I still want to obtain more single colonies. Plating a loopful of cells hasn't yielded enough single colonies for my needs, possibly because I don't spread the cells enough when streaking. Therefore, I'm considering redoing the electroporation with the plasmid, but this time starting with 5ng instead of 20 or 50ng. I believe this might help me achieve more single colonies.
Additionally, I will include your suggestion in my approach: Repeat with 50ng DNA and prepare 10-fold and 100-fold dilutions of my transformation mix before plating. This should help reduce cell density and make it easier to isolate single colonies. It's a good idea!
Thank you so much for your help :)
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