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I have a problem with a bacterial transformation of a yeast gene that I can not solve.

I isolated yeast DNA and did a PCR to get my product. I am using pCGCUm vector with a GFP construct. I digest both my plasmid and my product with SmaI and ClaI for 4h each. After that I purify the product from a gel and perform a ligation with high efficiency T4 ligase for 30 minutes at 22°C. Then I transform my electrocompetent cells with electroporation (2000V, 5ms), recover it in 0.5ml LB for 2h and plate it on LB-A plates.

First time I only did:

  • insert1
  • insert2
  • Empty vector

and it did not work (empty plates for insert and confluent growth (after 18h) of some weird colonies on EV).

I did the same ligation again and this time I included controls

This is what I did:

  • insert1
  • insert2
  • Empty vector
  • negative control (EK cells + electroporation)
  • positive control (EK cells + undigested vector)

Now I got:

  • insert1 (no colonies)
  • insert2 (big colonies a lot of them)
  • Empty vector (big colonies a lot of them)
  • negative control (EK cells + electroporation) (no colonies)
  • positive control (EK cells + undigested vector) (big colonies a lot of them)

The negative control suggests that my competent cells are ok, at least they are not resistant to AMP.

What confuses me is that even with positive control I get some weird big colonies - if I got nice transformants here I would assume that either digestion of ligation was not successful, but now I doubt that).

Another thing that is weird is that now I got that colonies also on my insert2 plate.

The scan image of big colonies looks like that: Empty vector plate

I have a question if anyone has had that kind of colonies? I was thinking of maybe having contaminated cuvettes for electroporation but I had them in 100% EtOH for 3 days and then dried in fume hood for 3 hours before electroporation.

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  • $\begingroup$ Some questions: 1) Can you describe the pCGCUm plasmid in more detail? antibiotics, presence of ccdB gene or MCS, etc. Can't find it on the web. 2) What are EK cells? 3) Where did you get your transformation protocol? From someone else in your lab who's had it work for them? I've been told not to let cells recover from electroporation for more than 1 hr because they may lose the plasmid during prolonged recovery, but if this has worked for a labmate then that's not an issue. 4) Have you repeated the 2nd expt (with the extra controls)? If it's contamination it might not reappear a second time. $\endgroup$ – A. Kennard Nov 25 '13 at 21:04
  • $\begingroup$ I agree those big colonies do not look normal. It's possible that your competent cells are contaminated with some strange strain. You could try plating serial dilutions of your competent cells (at least 10^6 fold dilution) until you can see individual colonies, and see if it's a mix of normal looking and weird colonies. Again, ask other people in the lab if they've had contamination issues--one time I struggled for a long time with low tranformation yields until I learned someone else was having the same problem, and we made a new batch of competent cells and were fine. $\endgroup$ – A. Kennard Nov 25 '13 at 21:35
  • $\begingroup$ Hey. Thanks for comments. pCGCum is 5.5kbp long. It has an ampicillin resistance and MCS long 53 nucleotides - SmaI on 8 and ClaI on 47. EK cells are electro competent cells(sorry I translated it from german). The protocol is standard protocol that has been used in our lab for long time now and has worked ok till problems appeared lately. At the moment it is only 2 people that were actually doing it and other one have the same problems with that colonies. $\endgroup$ – MartinK Nov 26 '13 at 13:01
  • $\begingroup$ We made a new batch of electro competent cells and alkaline phosphatase is new. Ligation kit was changed before my experiments. I will make new batch of chemical competent cells and try with those and hope to have some success. I will also do plasmid prep on insert2 plate where I have the colonies and try to digest it, maybe they are just "weird positives" - which would still not give me the answer but at least I will know something more. I will also plate different dilutions next time repeating exp.2 with our EKcells, EKcells from other lab and chemical competent cells. $\endgroup$ – MartinK Nov 26 '13 at 13:05
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I did try another transformation:

  • again with our electrocompetent cells
  • Cells form other lab
  • chemically competent cells I made

The good thing is that on chemically competent cells there is no weir colonies, however efficiency of transformation was very low, but still, I think it is ok - will confirm it in next day if I really got the right insert.

On the other hand my electroporation still did not clear out. I got colonies on both our cells and cells from other lab, however in much lower amount there. I will try to change the cuvettes and try to use different electroporator as this seems to be the only thing that could make problems - although I am still not convinced about that :)

Thank you for your help and I will report the progress so if somebody else will have that problem again he could maybe benefit from my findings.

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  • $\begingroup$ So, the answer is clear now. The contamination was in cuvettes. Some kind of bacteria that can survive 70 and 96% Ethanol, probably in form of spores. What I did is I putted cuvettes in LB medium for several hours so I got growing cells, than soaked them in 70% ethanol overnight and rinsed them with 96% EtOH for 2 hours. Than I added LB to cuvettes, "recovered it on 37 for 2 h and plated on LB plate. Plate was clean next day and also I did the normal transformation which worked ok! $\endgroup$ – MartinK Dec 10 '13 at 12:57
  • $\begingroup$ Do you re-use cuvettes? If you do so, you should at least incubate and wash them in 0.1M HCl to get rid of DNA contaminants before washing them with 1% SDS and getting rid of all the detergent afterwards with a lot of water. We stopped doing this and either use the cuvettes only once when we really have to do electroporesis and the rest of the time chemical competent cells work fine. $\endgroup$ – Chris♦ Dec 29 '13 at 11:06
  • $\begingroup$ Yes, we reuse the cuvettes and we only washed them with ethanol. Everybody in the lab were doing it that way, but with all the problems I had with cloning I will use chemically competent cells from now on. The efficiency is the same and I like it more. $\endgroup$ – MartinK Jan 8 '14 at 9:21

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