Hey everyone! I need to get into the habit of documenting this every day so sorry for my lack of posting. I have made a big note on my whiteboard at home to post every day now! So i’ll fill you in what i’ve been doing with Nikki recently…
Last monday morning me and sophia made some LB agar (2 lots of 400ml) and made up four 100ml bottles of solution 1. In the afternoon once the LB agar had been autoclaved I made up 50 plates of LB agar and put 20mg of ampicillin in the LB agar and made up all the agar plates.
On Wednesday me and Nikki had a chat about what we’ll be doing and needs to be done for the project and I went home and did research on the project.
On Thursday I made up competent cells (using the DH5a E. coli cells) competent cells basically take up extracellular DNA… Anyway I had to grow the cells by using a colorimeter with the optical density at 600nm. The cells then had to be incubated to around 0.35. Unfortunately I left them in the incubator for a bit too long and they grew up to the optical density (at 600nm) of 0.41! (I’ve learnt that mistake early on now!)
Then I had to transform the cells (transformation is the alteration of a cell from the direct uptake of exogenous DNA from its surroundings and taken up through the cell membrane). i used four different main plasmids which were: pOG4, pOG04, pOG4101 and 1029. I then spread plated the different plasmids onto the LB agar that i’d made previously and left them to grow overnight in the incubator.
On the friday I then did plasmid DNA extraction on the DH5a cells and did alkaline lysis on the E. coli cells. (Alkaline lysis is a method used in molecular biology to isolate plasmid DNA from small volumes of bacteria!)
Today I met Rosie and she’s doing a similar project to me but with the brayford pool water. We worked together on the samples I’d extracted last week (doing the alkaline lysis) and did gel electrophoresis. We made up a 1% agarose gel into 100ml of TA bufer. 20 micro-litres needed to be used for the plasmid samples (of which there were 9 samples – Large pOG4A, pOG 4B, pOG 4 C, pOG 4 D, pOG 4 SUB, 1029 A, 1029 B, 1029 C and 1029 D) and 2 micro-litres needed to be used for the plasmids compared to when we did the DNA ladder of which 1micro-litre Was used but 20 micro-litres for both different types of samples. (The 20 micro-litres were made up 3 micro-litres of loading buffer, 2 micro-litres of sybur green and 14 micro-litres of water). The agarose gel was then put into the microwave to boil it and then had to be left to cool down and then was poured into the base.
Once the gel had set, the samples then went into the centrifuge for 5 seconds and then had to be put into the wells that had been made. There were 16 wells in total and two had to be left at each side. This then had to be left for an hour and a half for the proteins to move down. When the electrophoresis had been on for over an hour and a half; the gel was then put into the UV visulaiser. Mike showed me how to use it (which is pretty ace!) but unfortunately the sybur green didn’t work so we had to put the gel didn’t in ethidium bromide and left it for 10 minutes.
Once this had been left we then put it under the UV visulaiser again and found out that only the DNA ladders were showing up. It was then suggested to me that when I did the alkaline lysis method last week that I didn’t leave the pellets that were formed long enough to dry (Which I am still miffed at myself for!). So I’ve had to start from scratch.
I then made up 9 broth cultures using the Large pOG4A, pOG 4B, pOG 4 C, pOG 4 D, pOG 4 SUB, 1029 A, 1029 B, 1029 C and 1029 D again and have left them in a incubator over night at 37 degrees. I will be doing alkaline lysis and transformations tomorrow and I will be doing right this time! 😀
Author: 
//