A Year in the Lab: Running New Bacterial Plugs and Preparing for Bradford Water Collection

Monday 7th April 2014

This week started with the preparation of my new plugs from last week, ready to continue perfecting PFGE settings. The plugs were incubated in 0.1 x wash buffer for 1 hour. This was then removed and replaced with 1 x NotI buffer for another hour with gentle agitation. This was again removed and replaced with 1 x NotI buffer and NotI restriction enzyme, incubating overnight at 37ºC.

IMG_5756

I spent the rest of the day continuing to work on preparation for my PCR, focusing specifically on preparing reagents and conducting a temperature gradient using my CTX-M Bla genes primers.

Tuesday 8th April 2014

Today began with collecting the bacterial plugs from incubation, removing the buffer and enzyme then incubating the plugs in 1 x wash buffer for an hour with agitation. This was then removed and replaced with 0.5 x TAE buffer. While they were incubating in TAE I prepared and poured a 1% agarose gel.IMG_5085

This gel was then loaded with the plugs and an appropriate PFGE ladder before running overnight using the following settings:

  • 20 hours
  • 1% agarose gel
  • 0.5x TAE
  • 14ºC
  • 40-100 switch intervals per second
  • 6 V/cm
  • 30 minute stain, 10 minute destain

I decided to run this first gel of the week using the same settings as my last most successful PFGE gel, in order to visulise whether this new batch of plugs contained a more appropriate amount of bacterial DNA.

I spent the rest of the day beginning to comprise a list of reagents and materials required for my next water collection in Bradford, after my Easter holiday. Including: Autoclaved Duran flasks, gloves, water collection rod, tryptone water (TW), lactose peptone water (LPW), LB broth, MLSA + CTX, nutrient agar and nutrient agar slopes.

Wednesday 9th April 2014

Once my PFGE gel had finished I stained it for 30 minutes, destaining for 10 minutes before viewing under UV. The resulting gel was very disappointing demonstrating very light bands for the Escherichia coli bacterial plugs and none at all for the ladder. Based on my experience I knew that there was definitely a problem with the run and not just with the new bacterial plugs. I therefore decided to dismantle and clean all components of the PFGE CHEF-DR II unit, as well as replacing all 0.5x TAE buffer used.

I then prepared and poured another 1% agarose gel. This gel was again loaded with the plugs and an appropriate PFGE ladder before running overnight using the same settings as yesterday:

  • 20 hours
  • 1% agarose gel
  • 0.5x TAE
  • 14ºC
  • 40-100 switch intervals per second
  • 6 V/cm
  • 30 minute stain, 10 minute destain

I spent the rest of the day preparing all the broths required for my next water collection: Tryptone water (TW), lactose peptone water (LPW), LB broth. These were all prepared as before, combining appropriate reagents with distilled water, adjusting the pH, decanting into glass universals before autoclaving. Finally I collected together gloves, water collection rod and autoclaved 2 empty Duran flasks.

Thursday 10th April 2014

Today started with removing my PFGE gel and staining it for 30 minutes. I decided to hold off destaining and just view it straight away under UV based on the lack of stained bands yesterday. The resulting gel was my one of my best so far, demonstrating very clear bands for the ladder as well as both Escherichia coli bacterial plugs (E.5 and ATCC 25922). When comparing the two bacterial plug banding patterns you can clearly see the differences in band size and amount, indicating that the samples are different strains.

PFGE Gel

Staining and band clarity was still a little dark/blurred in places indicating slightly too much staining/DNA. I decided to destain the gel for 10 minutes to see if I could improve this, however it made little difference when re visulised. For my next gel it would be interesting to see if using less plug and destaining for 15 minutes would improve the band clarity.

Once finished I continued preparing all the agar plates required for my next water collection:  MLSA + CTX and nutrient agar. These were all prepared as before, combining appropriate reagents with distilled water before autoclaving. When the agar had cooled enough to pour, CTX was added to the MLSA and then all plates were poured aseptically.

Friday 11th April 2014

I finished off the week by making nutrient agar slopes as before, combining appropriate reagents with distilled water, heating until combined, decanting into glass universals and autoclaving. Once autoclaved the universals were then sloped and until to cool allowing the formation a sloped surface for inoculation.

I then decided to test my previous extracted Escherichia coli bacterial plasmid DNA for purity using the NanoDrop2000 (ND). Nucleic acids absorb strongly in the UV region of the spectrum, showing a characteristic maxima peak at 260nm, while proteins are visible at 280nm. Conventional spectrometry requires a fairly large sample, using the ND allows testing of sample volumes as low as 0.5μl, which helps to avoid this drawback.

nanodrop2000spectrophotometerNanoDrop2000 from Thermoscientific (Bioresearch Online, 2013)

Once cleaned a 1μl drop of sample is pipetted onto the pedestal and tested using the appropriate software. The software calculates: DNA in ng/μl, A260, A280, 260/280 and 260/230. This can then be confirmed through manual calculation. Pure DNA has a reading of approximately 1.9, pure RNA approximately 2.1 and pure protein approximately 0.6. A reading of 1.75 indicates that there is approximately 50% DNA and 50% protein.

As I thought, my samples demonstrated mixed purity, ranging from very pure to inadequate. This highlights the main drawback of conducting plasmid isolation via alkaline lysis – variable purity. I hope that this problem will be rectified by using the QIAcube, which conducts plasmid isolation automatically and therefore improves purity.

Next week…

I will be spending the next week back home for the Easter holidays. When I get back I want to carry on finalising settings for my PFGE by using less plug to reduce the amount of DNA present on the gel and adding a smaller ladder to help determine the size of the smaller bands visible. Hopefully the QIAcube will also be fixed and I can then perform plasmid DNA extraction before beginning PCR. I also want to move onto collecting water samples from Bradford, processing these and looking specifically for antibiotic resistant Escherichia coli in order to create a similar collection to Lincoln. 

A Year in the Lab…

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One thought on “A Year in the Lab: Running New Bacterial Plugs and Preparing for Bradford Water Collection

  1. Pingback: A Year in the Lab: Finishing Bradford Bacterial Plug Production, PFGE and PCR | A Year in the Life...

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