A Year in the Lab: Modifying Bacterial Plugs and Plasmid Identification

Monday 28th April 2014

This week started with preparing my previously extracted Lincoln Escherichia coli collection plasmid DNA samples for running on gel electrophoresis. As I had a large amount of samples to run, I decided to spend today preparing all the samples for pipetting and then run them tomorrow. Extracted plasmid DNA from each sample was combined with loading buffer and pure water, then stored in the fridge. I also prepared a 1Kb ladder in the same way to use as a reference. I made enough to run alongside 8 gels, using 15 plasmid samples, as each gel tank and comb had 16 wells in total. Finally I prepared 1x TAE buffer, which would be used to make agarose gels, run the gel in and stain afterwards.

Tuesday 29th April 2014

I decided to run two gels at a time as I felt I would be able to prepare, pipette and run this amount without getting confused or making silly mistakes. I began by preparing and pouring 2 1% agarose gels before placing them into the tanks, filling them with 1x TAE buffer and loading them with the plasmid DNA I prepared yesterday. As I haven’t liquid pipetted into wells for a loooong time, this was fairly difficult the first few times! However, after 30 samples I felt much more confident.

IMG_6231

I ran both gels at 110V for 1 hour (As I was using small gel electrophoresis tanks), stained for 15 minutes and viewed under UV. I was very happy with my first two gels, as expected several samples demonstrated various different banding patterns while a few did not. I made a note of the samples which did demonstrate banding as this indicated that these samples contained plasmid DNA. This is interesting for my research as through PCR it can be determined whether this plasmid DNA contains specific genes (Bla genes and CTX-M) which code for the production of ESBL’s that play a direct role in antibiotic resistance to monobactams, penicillins and cephalosporins.

Wednesday 30th April 2014

Today began with starting new bacterial plug production, focusing on reducing the amount of DNA present in order to try and help resolve the problems seen last week. I inoculated one LB broth with a single colony from a sloped Escherichia coli sample from the Lincoln collection and another with the reference strain ATCC 25922. These broths were incubated, shaking at 37ºC for 4 hours until their OD reached 0.6-0.8. I chose to reduce the OD slightly from before in order to help reduce the amount of DNA present within the plug and therefore the darkness of the banding patterns. Each bacterial broth was then processed as before, centrifuging before combining with cell suspension buffer and agarose, pipetting into molds, incubating in lysozyme and then in proteinase K overnight at 50ºC.

IMG_6227

Once finished I then continued gel electrophoresis of pre prepared plasmid DNA samples. This was conducted in the same way and I was able to run 4 new gels throughout the day. As on Tuesday I was very happy with the gel images produced and I again noted down the samples which did demonstrate banding patterns.

Thursday 1st May 2014

Once the plugs had finished incubation in proteinase K, each plug was washed to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs were then stored in 1x wash buffer at 4ºC.

I then finished off gel electrophoresis of my last pre prepared plasmid DNA samples. This was conducted in the same way as before and I was very pleased with the gel images produced from my final 2 gels.

PFGE Gel 4

Friday 2nd May 2014

I decided to spend Friday printing off copies of all the gel images from the week, identifying plasmid DNA banding patterns as well as labeling ladders and Lincoln collection samples. This allowed me to produce a list of samples which I will be carrying on to investigate through PCR, due to the presence of plasmid DNA.

Next week…

As my new CHEF DNA Size Standard Lambda Ladder has arrived and my new batch of bacterial plugs are ready I can see whether the reduction in DNA has been successful and use the new ladder to begin approximating the smaller bands visible. I had also hoped to be starting on PCR, however my Taq Mastermix and 50bp ladder haven’t arrived yet so I will have to wait and keep planning until they get here!

I will also FINALLY be collecting water samples from Bradford next Friday – which is very exciting as I can’t wait to begin a comparable Bradford collection of Escherichia coli isolates. The journey up to Bradford should take around 2-3 hours, there and back, so it’s looking like it will be a long day and an even longer weekend…

A Year in the Lab…

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