Transformation problems - time of transformation very low (0.7 ms) ???? (Jan/10/2007 )

Hello everybody,

I'm preparing 16 S rDNA libraries specific of the Archae domain. We usually use a TA cloning system (pGEM-T Promega) and then a transformation with electrocompetent cells (DH10B) and finally a White/Blue selection.

During the ligation, we are using a ratio of insert : vector of 3:1 for Bacteria libraries but with Archae as I had more than 80 % of blue colonies at the end, we tried to increase this ratio to approximately 8:1. But during the transformation process, using the same electroporation cuves the same voltage and the same competent cells the time given by the electroporator was around 0.7 ms instead of 4-5 ms.

I don't know the origin of the problem ?? The time of transformation is related to what ??? Do you have an idea ?
Thanks for your suggestions

Delphine
France

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My guess would be that you had electric arcs... Did you notice a sharp noise when you did your electropration? Arcs are usually due to too much salts, and that can be link to the amount of DNA you're using, the state of your cuvettes (if they're not clean enough for instance), the concentration of cells and the buffer you're using...
Anyway, an arc would kill your cells, so you shouldn't get any transformant on your plates if that's the case, and as I said, it's quite noisy...

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I forgot to precise that there was no electric arcs... That's the mystery...

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0.7ms, no electric shock sufficent for transformation...
did you try a new cuvette?...

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It did the same thing with 7 samples i.e. 7 different cuvettes

But I think maybe there was not enough vector for the transformation because of the new ratio insert : vector the vector was diluted and finally there was only 1ng of vector instead of 10 for the transformation ... I did another ligation with a larger final volume and I will put 10 µL of ligation instead of 2 as we usually do in order to reach the same 10 ng of vector but with a ratio of 8:1.

I will tell you if it's better...

thanks everybody for your help

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Hello

Well I have had this problem as well. I think you have too much salt in your ligation and probably you were very close to get an eletric arc.
Anyway I think 0.7 ms might not be enough for the efficient incorporation of plasmid in the cells.
What I did: I diluted the bacteria plus ligation with ddwater (you can add +-300ml water to the cuv) I got much better values with this approach (4ms) and nice (more important colonies carryng the plasmid with insert) the next day.

AND DONT WORRY, IF YOU GET AN ELETRIC ARC YOUR CELLS ARE NOT DEAD. YOU ONLY NEED TO ELETROPORATE IT AGAIN UNTIL YOU GET 3-5ms (AS I SAID, ADD WATER). PEOPLE THINK THEY ARE DEAD ´CAUSE THEY DONT ELETROPORATE AGAIN (ELETRIC ARC=NO ELETROPORATION=NO PLASMID CARRYNG RESISTENCE MARKER IN THE CELLS) AND THEREFORE DONT GET ANY COLONY IN THE NEXT DAY (NO PLASMID COULD ENTER THE CELLS).
I hope I helped!

Adriana

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Salut Delphine,

For me it looks like when you changed your ligation ratio, somehow the salt content has changed as well, hence the electroporation problem. I had this happpening before. I solved it by diluting the ligation with 10% glycerol/ddwater (that's the media we use to rinse the bacteria when we prepare electocompetent cells). Of course that implies that your DNA concentration in the ligation reaction can be diluted so that you're still in the range of acceptable amount for electroporation... Actually I do that for each tricky ligation I do now. I never get any arc or short ms after implementing this trick in the electroporation protocole.
The second thing is how you recycle your cuvettes. I treat them with 5% bleach and rinse them 3 times with ultrapure water then I rinse with 95% ethanol and let them dry in a 80C oven. I eliminated the troubles we encontered with the recycled cuvette doing this.

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according to the manual of the eletroporator we use, you can only use cells that arced maximum 3 times.

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