Tag: plasmids

YAY got growth finally can start I took…

Deborah / / Uncategorized

YAY!! got growth, finally can start. I took four colonies from each of my culture plates and made them up into broths using LB broth and adding ampiillin 100. Then i placed them in the shaker at 37C, until they grew to an OD of 1(0.8-1). then i set up a dilution series, labelling eppendorfs A,BC,D -1 to -5 adding 0.9ml NaCl and 0.684ml into the eppendorf marked -5.5. Once the broths reached an optical density of 1, 0.1ml was added to each eppendorfs with the exception of the -5.5 as 0.316ml broth was added. From the 10 -4 eppendorf, 100microlitres was innoculated into 10ml of LB broth and incubated overnight at 37C. The -5 and -5.5 dilution series were spread plated onto LB agar plates and incubated overnight at 37C. This was repeated for pOG04 NJC and pOG04 DRW plasmids. Cant wait for tomorrow.

Hey Started the day by making 400ml of…

Deborah / / Uncategorized

Hey,
Started the day by making 400ml of LB agar and 200ml with AMP100 and 500ml of LB broth. Then i counted the T0 replica plates i did yesterday. Next i counted yesterdays T end plates and chose the ones with approx. 100 colonies and replica plated them onto LB agar and LB agar with AMP100. Then went to the presentation for poster presentation and project write up. Then i streaked a fresh LB agar plate with C2110 colony and then another LB agar plate with DH5alpha (for Rosie) and incubated them at 37C overnight, so that we have fresh colonies to work with next week. Nicola gave me my plasmids, popped them in the fridge. Nearly all prepped for next week. Also managed to make up stabs and slopes of E.coli e0791 that i brothed up yesterday. Also BIG thanks to Dan for his anthropology presentation and showing me the difference between boys and girls (on the skeletons). Very interesting seeing the ossicles and learning how to age and sex the bones.

Hello Today i started producing competant cells and…

Rosie Myers / / Uncategorized

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! :):)

Hey all I made some competent DH5 alpha…

Soph Marie Scanlon / / Uncategorized

Hey all!
I made some competent DH5 alpha E. coli cells today (which took 5 and a half hours to grow to the optical density of 0.350!) and used the colorimeter to see if they had reached the optical density . I have also transformed them so I can do alkaline lysis tomorrow! Still in the lab currently waiting as the transformed cells are currently in the incubator for 45 minutes then I need to spread plate them on to LB agar plates that have ampicillin in them and put them in a incubator over night. I’m using the same plasmids again (pALA1029, pOG4, pOG04, pOG4.003).
See you all tomorrow!
Soph 🙂