Monday 30th June 2014
As I am moving out of my flat in Lincoln this week, research work had to take a bit of a back seat! I started the week still in Lincoln, preparing to run all plasmid positive samples through my established PCR. I selected the samples I wanted to process then calculated the amount of PCR tubes, positive/negative controls and reagents I will need. Once these were prepared I calculated the number of gels I will need to run in order to visualise all the samples. I then gathered together gel electrophoresis tanks and gel boxes, before ensuring I had enough 1x TAE buffer, loading buffer and 50bp ladder. I autoclaved my prepared buffer and chilled it in the fridge prior to use.
Thursday 3rd July 2014
Today began with the preparation of all PCR reactions within PCR tubes. I decided to run two separate PCRs, one for all Lincoln samples (Including a positive and negative control) and another for all Bradford samples (Again, including a positive and negative control). To ensure accuracy I prepared tables for each, which demonstrated the exact position of each sample within a 8 (A, B, C, D, E, F, G, H) by 12 grid. As before I used NCTC 13353 as my positive control and ATCC 25922 as my negative control.
I started with my Lincoln samples and carefully pipetted the required reagents into 50μl PCR tubes, including mastermix Taq2x, all multiplex group primers (1 in 10 dilution), plasmid DNA (1 in 1000 dilution) and pure water. I then placed this into the PCR machine, and using the settings I have established from previous practice PCRs ran for 2 hours. I then repeated this process for the second PCR, using all my Bradford samples.
Once both PCRs had finished it was quite late so I decided to store the PCR product overnight in the fridge before running the samples tomorrow.
Friday 4th July 2014
Friday started off with a supervisor meeting in which we discussed both mine and other postgraduate’s research progression over the last month, as well as changes that will be occurring within the College of Science (Including our move to Joseph Banks Laboratory, which unfortunately I will just be missing!).
Once finished I spent the rest of the day running the resulting PCR product for both the Lincoln and Bradford samples on 1.8% agarose gels for 1.5 hours, alongside 50 bp ladders for reference. I stained the gels for 15 minutes before viewing under UV. The resulting gels were very disappointing, demonstrating clear and accurate banding for the 50bp ladder but not a single band for any of the samples. The negative control showed no banding and the positive control showed a clear band approximately 415bp in size which confirmed that my PCR had worked correctly. As a result of this the lack of any sample bands either means that my sample DNA was too diluted (1 in 1000) or I don’t not have any samples which contain ESBL producing CTX-M Bla genes.
Unfortunately I ran out of time to run and visualise my last two gels, or re perform PCR with adjusted settings so I decided to put research work on hold until I am back in a weeks time.
As I have now officially moved out of my flat in Lincoln, I will be spending the next week back in Oxfordshire completing a few days of induction for my new job which starts in September 🙂 I will then be returning to Lincoln and staying at a friend’s house for a week in order to finish off my final research work before focusing entirely on writing up my thesis. The plan for my final week is to finish running my last 3 PFGE gels and input the resulting gel images into Gel Compar software before conducting comparative analysis during August. I will also be running and visualising my last two PCR gels before re performing with adjusted DNA concentration/dilution after Nanodrop testing.
A Year in the Lab…