A Year in the Lab: Bradford Sample Plasmid Extraction and Gel Compar Training

Monday 12th May 2014

This week continued from my water processing work at the weekend, with observing inoculated lactose peptone water (LPW), tryptone water (TW) broths and antibiotic susceptibility agar plates. Colour change of LPW was recorded (Positive – red to yellow/Negative – no colour change) and Kovac’s reagent was added to TW before observing any colour change (Positive – cherry-red coloured ring/Negative – no colour change). Zone clearance (to the nearest mm) for the incubated mueller hinton antibiotic susceptibility plates was also measured and recorded within a prepared table in my lab book.

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I then began preparing LB broth, ready for inoculation with isolated Bradford samples for plasmid extraction in the QIAcube. This was done by combining broth powder with distilled water, decanting into glass universals and autoclaving. Once finished and cooled these were inoculated with isolated Bradford samples, from agar slopes, and incubated overnight shaken at 37°C. I also decided to make some nutrient agar plates which will be streak plated with all isolated Bradford samples to ensure they are all only pure Escherichia coli.

Finally, I prepared all the equipment required for plasmid extraction tomorrow with the QIAcube. This involved autoclaving eppendorfs for the final plasmid DNA, setting up rotor adapters and labeling up 2ml eppendorfs with sample numbers.

Tuesday 13th May 2014

I started the day by removing my inoculated LB broth, pipetting 2ml of each into individual prepared eppendorfs before centrifuging them for 5 minutes at full speed. While waiting, I placed the pre prepared rotary adapters into the QIAcube internal centrifuge, re filled pipette tips and reagents. Once the samples had finished centrifuging I tipped away the supernatant and placed the 2ml eppendorf containing the remaining cell pellet into the QIAcube.

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Each run of 12 samples took around 50 minutes, and I managed to get samples 1-48 completed with no problems. The extracted plasmid DNA for all samples were stored in the fridge until later in the week when these samples can be run on gel electrophoresis in order to visulise if plasmids are present. I decided to finish off the rest of the Bradford Escherichia coli isolates tomorrow.

Wednesday 14th May 2014

Today began with the continuation of Bradford isolate plasmid extraction, for samples 49-100. Once completed, the extracted DNA for all samples were stored in the fridge, as before.

Throughout the day I also prepared reagents and equipment ready for gel electrophoresis. I prepared 5L of 1x TAE buffer, by combining 50x TAE stock with distilled water before autoclaving to ensure very clean buffer.

I spent the rest of the day in a training session with the laboratory technician for molecular biology, regarding the gel comparison software – Gel Compar. Myself and another postgraduate student were fortunate enough to be able to have a one on one session on how the software works, as well as running through the process step by step, from uploading gel images to producing dendrograms. Although I am not quite ready to be comparing my gels, this software will be very useful in the next few weeks as I begin producing bacterial plugs for all samples within both my Lincoln and Bradford collections, and running through PFGE to analyse strain differences/similarities.

Gel ComparGel compar Software (Biosystematica, 2013)

Thursday 15th May 2014

As I have a large amount of samples to run, I decided to spend today preparing all the samples for pipetting, running them tomorrow and Saturday. 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 7 gels, using 15 plasmid samples, as each gel tank and comb had 16 wells in total.

Finally I placed my pre prepared 1x TAE in the fridge to try and keep the running temperature of the gels as low as possible and prevent blurring as a result of excess heat. I also set up 4 gel tanks and power packs on my bench, as well as taping up 4 gel boxes in preparation for tomorrow.

Friday 16th May 2014

Today began with preparing 4 1% agarose gels, by combining agarose powder with 1x TAE buffer before heating until homogeneous. Once cooled slightly, agarose was then poured into the pre prepared gel boxes and allowed to solidify. While waiting I retrieved my pre prepared Bradford plasmid DNA samples from the fridge, and gathered together an appropriate pipette and tips. I placed my prepared 1% agarose gels into the tanks, filled them with 1x TAE buffer and then loaded two at a time with the pre prepared plasmid DNA samples, as well as the 1kb ladder (Samples 1-60).

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I ran the gels at 110V for 1 hour and 30 minutes, stained for 20 minutes and viewed under UV. I was very pleased with my first four gels, as expected several samples demonstrated various different banding patterns while others did not. I made a note of the samples which did demonstrate banding as this indicated that these samples contained plasmid DNA. This is useful 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.

Saturday 17th May 2014

I spent today finishing off gel electrophoresis for my plasmid DNA samples 61-100. This was conducted in the same way as yesterday and I was able to run my final 3 gels throughout the day. As on Friday I was very happy with the gel images produced and I again noted down the samples which demonstrated banding patterns.

PFGE Gel 4

Finally, I printed off copies of all the gel images from the week, identifying plasmid DNA banding patterns as well as labeling ladders and Bradford collection samples. This allowed me to finish off my list of samples which I will be carrying on to investigate through PCR with my already identified Lincoln samples, due to the presence of plasmid DNA.

Next week…

For the next two weeks my time in the lab will be fairly limited as I will be working as an exam facilitator for students at the University of Lincoln. Although I have been working as a note taker and facilitator since October 2013, I haven’t had the opportunity to work as an exam reader/scribe. I have been assigned to work with 5 different students who will be taking exams in a variety of subjects which should be really interesting 🙂

Once finished I will be continuing my research work by producing bacterial plugs for all Lincoln/Bradford samples identified as having plasmid DNA present, and begin analysing these through PFGE and gel compar software. I will also be optimising my individual PCR primers (Which are part of a multiplex) before again processing all Lincoln/Bradford samples identified as having plasmid DNA present.

A Year in the Lab…

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