A Year in the Lab: PFGE and Glycerol Stocking

Monday 10th March 2014

This week started off by completing plasmid isolation on my remaining Lincoln sample collection. I inoculated LB + CTX broth with a single colony from my collection of Escherichia coli nutrient agar slopes (E50-.140). These were then incubated overnight with agitation at 37ºC.

Once finished I then prepared more plugs 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.

Tuesday 11th March 2014

Today began by removing my inoculated LB + CTX broths and isolating plasmid DNA through alkaline lysis. Once completed, the plasmid DNA was stored at 4ºC until tomorrow when it will be run through gel electrophoresis to visulise banding patterns. I also inoculated tryptone water (TW) and lactose peptone water (LPW) with a small amount of the overnight LB + CTX broths before incubating overnight at 37ºC.

I collected the bacterial plugs from incubation, removed the buffer and enzyme then incubated 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. 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 keep all the settings the same as for my last gel, except for the switch intervals which I reduced slightly in order to clean and tighten up the bands that are have become more visible on my previous PFGE gels.

I then began glycerol stocking my Lincoln bacterial isolate collection by initially streak plating sloped isolates (E.1-41) onto nutrient agar in order to ensure all samples are purely Escherichia coli and to achieve single colonies. These plates were then incubated overnight at 37ºC. Fortunately I already have some LB broth and 10% glycerol which was given to me by another student in the lab, so I have enough to continue glycerol stocking tomorrow. Instead of using flip top eppendorfs I will be using screw top, therefore I also prepared and autoclaved screw top eppendorf tubes separately.

IMG_5648

Finally, I made and autoclaved more nutrient agar slopes, before finishing re sloping my bacterial collection.

Wednesday 12th March 2014

I started off today by running the plasmid DNA samples which I isolated the day before on gel electrophoresis in order to visulise the banding patterns present. The resulting gel demonstrated plasmid banding patterns between 1.0 and 1.5 kb for samples: 53, .53, .92 and .126. This therefore indicated that these Escherichia coli samples contained isolated plasmids which can then be run through PCR to look for CTX-M specific Bla genes.

Once my PFGE gel had finished I stained it for 30 minutes, destaining for 10 minutes before viewing under UV. The resulting gel demonstrated good clear individual bands for 2 of my bacterial isolate samples (52 and 53).

PFGE

For sample 52 – 2 very clear bands approximately 680kb and 610kb, and 2 blurred bands approximately 356-450kb and 285-365kb. For sample 53 – 2 very clear bands approximately 945kb and 610kb, and 2 blurred bands approximately 356-450kb and 285-365kb. The next step I would like to pursue is producing a further batch of bacterial plugs using samples from my new Lincoln collection, in order to perfect my PFGE running procedure.

I then removed my streak plates from the incubator and inoculated LB broth with a single colony from each plate. These broths were incubated overnight at 37ºC.

IMG_5687

Finally I removed the overnight LPW and TW inoculated with E50 -.140. I added Kovac’s reagent to the TWs then observed and noted down the results to both tests. A colour change from red to yellow in LPW and the presence of a cherry red ring in TW, indicate Escherichia coli.

Thursday 13th – Friday 14th March 2014

Today began with the removal of my overnight LB broth cultures and continuation of glycerol stocking. I pipetted 500μl of each overnight LB broth culture into separate tubes before adding 500μl of 10% glycerol LB broth. These were then carefully labelled and stored in the freezer.

IMG_5645

Next week…

Now I have perfected PFGE settings for the current batch of bacterial plugs I have been using, I would really like to move onto running further PFGE gels using E.coli samples from my new Lincoln collection. I would also like to finish off glycerol stocking my collection to ensure I have continued access to my isolates over the next few months. As I have selectively identified CTX resistant/intermediate E.coli, isolated and visulised plasmids I have significantly reduced my number of original isolates. Therefore I would like to collect another water sample in order to increase my number of samples a bit more before starting to conduct PCR.

A Year in the Lab…

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4 thoughts on “A Year in the Lab: PFGE and Glycerol Stocking

  1. Hi Amy

    Thanks for the blog – great detail. Have you thought if the not1 is discriminatory enough for your strains (isolates) Do you need to try other enzymes What does the literature say? Also have you thought you should transfer these bands to nylon or nitrocellulose to 1) preserve them 2) probe them with antibiotic resistance genes?

    Hope this helps. The boss

    • Thank you very much 🙂 The main piece of literature (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1753.pdf) I have been working from suggested using NotI and SfiI so I decided to start with NotI, however it would be really interesting to try out SfiI too once my plug production and running protocol are perfected. No, I haven’t thought about that – would that be along the lines of southern blotting? If so that sounds like it would be something good to try, especially in terms of antibiotic resistance gene analysis along with PCR. Thank you for the advice!

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