A Year in the Lab: Lincoln Bacterial Plug Production, PFGE and PCR

Monday 2nd June 2014

This week began with conducting a final PFGE run with both my PFG and lambda ladder, one Escherichia coli sample and reference strain ATCC 25922 to confirm settings before mass plug production and running. As previously 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.

I then finished off my preparation for PCR by re-suspending my primers using pure filtered water to produce a 100μM stock, before diluting further to a 10μM working stock. I also diluted my practise bacterial DNA sample in the same way and used to Nanodrop to confirm the presence of DNA before use with PCR.

IMG_6435

Finally I set about calculating the amount of reagents required to produce a large batch of plugs, as I have only ever produced around 10 plugs at a time using 1/2 samples. Once finished I gathered together and prepared the reagents, including: LB broths, 2% agarose, cell suspension buffer, chloramphenicol, lysozyme and proteinase K.

Tuesday 3rd June 2014

I started today by inoculating my prepared LB broth with single colonies from my Lincoln agar slope collection. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm. If the sample was above 0.7 abs then chloramphenicol was added to inhibit replication and the sample was left out on the bench. However, if the sample was below 0.7 abs it was returned to the incubator for another hour to allow for further growth to occur. Once reached, each LB broth culture was 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.

Once finished I collected my bacterial plugs from incubation, removing the buffer and enzyme before 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.

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This gel was then loaded with the plugs, PFG and Lambda ladders 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 ran this gel using the same settings as my last most successful PFGE gel, in order to confirm that these settings are the most appropriate to visulise whole genome banding patters for Escherichia coli. This is in preparation for mass plug production (Of all samples identified as being Escherichia coli, antibiotic resistant and demonstrating plasmid DNA banding when ran through standard gel electrophoresis) and running through PFGE, in order to analyse banding patterns in relation to strain analysis.   

Wednesday 4th June 2014

Today began with the washing of my bacterial plugs, in order to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs stored in 1x wash buffer at 4ºC until more restriction enzyme arrives in the lab.

IMG_5051

Once my PFGE gel had finished I stained it for 30 minutes, destaining for 10 minutes before viewing under UV. The resulting gel was consistent with the previously gel and confirmed that these settings are the most appropriate I have developed for running bacterial plugs containing Escherichia coli samples, alongside PFG and Lambda ladders. Now I have finalised these settings I can continue with mass plug production for all samples within my Lincoln and Bradford collections which fit my desired criteria, before running in batches through PFGE.

I spent the rest of the day preparing, running and visulising my first attempt at PCR. I decided to begin with just my CTX-M group 1 primers, using my previously extracted plasmid DNA as a positive control (Confirming DNA presence using the Nanodrop) and replacing this for water in a negative control. I carefully pipetted my required reagents into 50μl PCR tubes, including mastermix Taq2x, primers, plasmid DNA and pure water. I then placed this into the PCR machine and conducted a temperature gradient, ranging from 50ºC to 63.8ºC in order to optimise the most suitable temperature for group 1 forward and reverse primers.

IMG_6442

After 2 hours my PCR finished and I ran the resulting PCR product on a 1.8% agarose gel for 1.5 hours, alongside a 50 bp ladder for reference. I stained the gel for 15 minutes before viewing under UV. The resulting gel was disappointing but not surprising, my ladder was present and demonstrated clear bands however my plasmid DNA was not suitable as a positive control. I had previously expected that this may be an issue. so for my next attempt at PCR I will be using the reference strain Escherichia coli NCTC 13353 instead of using plasmid DNA. NCTC 13353 is recommended for use as a positive control for CTX-M group 1 ESBL’s, and it is currently glycerol stocked in one of the lab’s freezers.

Thursday 5th June 2014

As on Tuesday, today started by inoculating LB broth with single colonies from my Lincoln agar slope collection. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm. Once reached, each LB broth culture was 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.

I spent some time looking through the reference strains that we have in the lab’s freezers, and found 5 that could be useful for my particular research area. NCTC 11560, which is a TEM-1 Beta lactamase producer. NCTC 13353 (that I mentioned previously), which is a CTX-M-15 (Cefotaxime) ESBL producer and is often used as a control strain for group 1 bla CTX-M multiplex PCR assays (Like mine!). ATCC 23355, which produces cephalosporinase beta-lactamase II and can act as a bacteriophage host. ATCC 7006003A/B, which are SHV-18 producers. In order to check whether these glycerol stocks were still viable and visulise single colonies I decided to streak and spread plate them onto nutrient agar, along with ATCC 25922 which I use as a reference for identification of Escherichia coli. These plates were then incubated overnight at 37ºC.

Friday 6th June 2014

As on Wednesday, I spent today washing the bacterial plugs, in order to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs stored in 1x wash buffer at 4ºC.

Out of the 6 reference strain nutrient agar plates NCTC 11560 was the only sample that didn’t grow. All others showed good growth on the spread plate and clean single colonies on the streak plate. In order to perform DNA extraction of the reference strains I then inoculated each into separate LB broths. These broths were then incubated shaken overnight at 37ºC.

IMG_6471

Saturday 7th June 2014

I started today by inoculating LB broth with single colonies from the last of my Lincoln agar slope collection. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm. Once reached, each LB broth culture was 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.

I had hoped to use my overnight LB broth cultures in the QIAcube, however when I arrived and tried to turn it on the screen displayed an error which indicated that a software issue was preventing me from accessing any of the programmes. I emailed our laboratory technician to let him know, but as it was a Saturday I knew there was no way I was going to be able to do an extraction today. So, I decided to centrifuge the samples anyway, remove the supernatent and freeze the pellet for use in the future.

Sunday 8th June

I finished off this week with bacterial plug washing! The washing procedure and times were kept the same as previously, and plugs stored in 1x wash buffer at 4ºC.

Next week…

Monday afternoon I will be traveling back home to Oxfordshire to attend job interviews for when I finish up my masters in September! Depending on what happens at the interviews I will be away for most of next week, and so lab work will be on hold until I’m back. When I get back I will be carrying on with bacterial plug production, beginning the Bradford part of my collection now all my Lincoln plugs are made. Hopefully more restriction enzyme will have been delivered and I can get started on restriction digesting the new plugs ready for running through PFGE. Lastly if the QIAcube is not up and running again I will be manually extracting DNA from my reference strains and having another go at PCR!

A Year in the Lab…

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