Make sure you are using the correct width of cuvette. They come in 4mm, 2mm, and 1 mm width, and you need to use the same as your paper does.
I am using the 1mm as they do it in this protocol, so it should not be a problem.
Also, pay careful attention to how they prepare the cells. What phase of growth do they harvest at? How are they washed?
The protocol for preparing electrocompetent cells is quiet long and tedious. In short: you have to grow O/N culture in BHI, than dilute this suspension to 10% in BHI medium+ 0.5M sucrose (BHIS). Grow till one stage. Add penicillium. Grow till the OD doubles and then cfg and wash with descending volumes of cold sucrose-glycerol wash buffer (SGWB) for several times. add also in one step lysozime. then cfg and wash again with SGWB and cfg and wash with SGWB. aliquot the suspension in 50uL volumes and store at -80 degrees. Everything on ice all the time.
I would do a viable colony count on the prepared cells both before and after electroporation by plating out serial dilutions on non-selective plates.
I am always doing a negative control (bacteria+ water) and plating on non-selective plates 3rd and 5th dilution and bacteria grow nicely (the plate is full of bacteria, so I never counted the Nu.). But I will try that to see the exact viable colony count. Thank you for this note
How are you recovering the cells? Do you grow them with non-selective medium for an hour or more, followed by plating on selective plates? What do your co-workers do?
I am recovering them as they said in protocol (actually the protocol is very, very detailed for an article with lots of notes, but it still does not work
() : with 1mL room temperature BHIS, statically at 30 degrees in incubator for 1,5h. Do the serial dilutions with BHIS and plate them on selective plates with concentration of selective antibiotic as the people from this other lab, from where I have got the plasmid, have recommended to me.
I am the only person in my lab doing cloning stuff, also the youngest one and I have asked the authors quiet a lot of questions already and I really stick to their advices, but still no result. That is why I asked here, if somebody has some extra advice. Thanks for all your comments and notes. It is really good to go with somebody else through the protocol again and check the crucial points of it.
Worst case, can you visit the lab that did these experiments and watch how they do it?
Well, maybe I could be able to arrange that on my own cost during my holidays
Thank you once again