A Year in the Lab: Continuing Plasmid Isolation and PFGE

Monday 3rd March 2014

Today began with stock preparation for the week. I prepared fresh LPW (Lactose peptone water), TW (Tryptone water), LB + CTX broth, Mueller Hinton agar and MLS + CTX agar. As I have made all of these before I used previously written methods and calculations, then autoclaved before storing at 4ºC.

Once finished I prepared my newest batch of bacterial plugs for PFGE by first incubating them 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 4th March 2014

I started the day by 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 I prepared a 1% agarose gel as before. I then loaded this gel with the plugs as well as an appropriate ladder before running overnight using the following settings:

  • 22 hours
  • 1% agarose gel
  • 0.5x TAE
  • 14ºC
  • 50-110 switch intervals per second
  • 6 V/cm
  • 10 minute stain, 10 minute destain

Based on my last PFGE gel I have reduced the run time, to prevent the sample DNA running off the bottom of the gel. As these are new plugs I will be using switch intervals, temperature, gel percentage and voltage settings I know work well for Escherichia coli.

Once finished I then added CTX antibiotic to the LB broth I made yesterday, before inoculating each with a single colony from my collection of Escherichia coli nutrient slopes (E1-24). These were then incubated overnight with agitation at 37ºC.

IMG_5563

Wednesday 5th March 2014

Today began with removing my overnight PFGE gel, staining for 10 minutes then destaining for 10 minutes before viewing under UV. The resulting gel was very light and therefore it was decided to increase the staining time to 20 minutes with a 10 minute destain to help increase the visibility of the DNA present. Unfortunately the run time was too long, and this meant that the samples ran off the bottom of the gel. It was therefore decided to reduce the run time to 20 hours. Despite this the presence of a few bands could be seen within the blurred sample lanes and hopefully will be much clearer once run time and staining have been modified.

AL 10001

I prepared and loaded a new gel, running this with the modified settings overnight:

  • 20 hours
  • 1% agarose gel
  • 0.5x TAE
  • 14ºC
  • 50-110 switch intervals per second
  • 6 V/cm
  • 20 minute stain, 10 minute destain

Once finished I removed the incubated LB and CTX broths, and inoculated each into both LPW and TW before incubating overnight at 37ºC. I then began conducting plasmid isolation through alkaline lysis on LB and CTX broth cultures E1-24. I did this the same way as last week, finishing off by running the resulting DNA samples through gel electrophoresis to visulise the banding patterns present.

IMG_5583

The resulting gels demonstrated plasmid banding patterns between 1.0 and 1.5 kb for samples: 4, 5, 6, 8, 9, 11, 12, 16, 18, 19, 21, 24 and 25. 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.

AL 10003

In preparation for further alkaline lysis tomorrow, I then inoculated more LB and CTX broths with single colonies from my collection of Escherichia coli nutrient slopes (E26-49). These were again incubated overnight with agitation at 37ºC.

Thursday 6th March 2014

I started today by removing the overnight LPW and TW inoculated with E1-25. 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.

IMG_5597

Once my PFGE gel had finished I stained it for 20 minutes, destaining for 10 minutes before viewing under UV. The resulting gel demonstrated that this modified staining procedure produced more visible band patterns, for both the ladder and samples. In order to improve this further I decided to increase the staining to 30 minutes, keeping the destain at 10 minutes. The change in run time worked very well and prevented the samples from running off the bottom of the gel. Samples are still demonstrating faint band patterns within the blurring, therefore in order to help clear and tighten up these bands I will be reducing the switch intervals times.

AL 20002

I then continued plasmid isolation on samples E26-49 using the same procedure as Wednesday. Again, when finished I ran the resulting DNA samples through gel electrophoresis to visulise the banding patterns present. The resulting gels demonstrated plasmid banding patterns between 1.0 and 1.5 kb for samples: 26, 27, 28, 31, 36, 38, 39, 41, 42, 48 and 49.

Finally I inoculated each LB and CTX broth culture into both LPW and TW before incubating overnight at 37ºC.

Friday 7th March 2014

Friday began with a supervisor meeting from 10-12pm. Once finished I removed the overnight LPW and TW inoculated with E26-49. As before, I added Kovac’s reagent to the TWs then observed and noted down the results to both tests. To my surprise the CTX-M primers I ordered on Tuesday arrived! So I decided to spend some time reading through the supplied information, then used the rest of the day to complete other work and plan for next week.

IMG_5599

Next Week…

I still have a few more bacterial samples to isolate plasmids from (The trouble with having such a large collection!), so I will continue alkaline lysis on these final samples before conducting transformations with my prepared competent cells and finally beginning preparation for PCR. As I have never run my own PCR before, I need to reacquaint myself with the equipment and reagents I will be using before I start.

I will also be running another PFGE gel using modified switch interval settings, in attempts to clear up the bands visible from samples. In the coming weeks it would be great to make another batch of bacterial plugs using some new samples from my established Escherichia coli isolate collection.

Finally, I would really like to get the whole bacterial collection re sloped and glycerol stocked, now I know for certain that all the samples are Escherichia coli and demonstrate CTX intermediate/resistance, as well as beginning to see which contain plasmids.

A Year in the Lab…

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