Hello!
Today i started producing competant cells and doing transformation.
Competent cells were made by first creating an LB broth by placing a colonie from a DH5 alpha plate into it, and having it incubated and shaking for around 5 hours! Once my cells were at the right optical density, I could proceed. As it happens they grew really fast and well so thats always a good start! Once I had centrifuged my cells down multiple times, placed them on ice, and added amounts of CaCl2 i ended up with cells which were then competent and capable of taking up plasmid DNA. The competent cells could be used immediately for transformation and (hopefully) uptake my plasmid DNA which i have obtained from my previous weeks in the lab!
I then began transformation where i added my different plasmid DNAs with some competant cells and proceeded to heat, cool, heat, cool etc! It is a very long process! But it enables the cells to recover and the antibiotic resistance of the plasmid to express itself 🙂
Basically with the solutions i then had, I spread 100ul from each one onto LB agar containing ampicillin, and if there are colonies present tomorrow morning then this means that the cells have uptaken my plasmids, and that i do have plasmids stocks that are resistant to amicillin! If i do i will have lots of things to play with 🙂 If not… i shall start all over again. 🙁
I have also spread a positive control, so that if it doesnt work tomorrow this will tell me if it was something in my methodology that was wrong or just that the plasmids in my DNA samples are not resistant to ampicillin, and it was probably just the chromosomes 🙁
Any questions feel free to come and ask! :):)
