A Year in the Lab: My Final Week in the Lab!

Monday 14th July 2014

This week began with preparing one of the final batches of plugs for running tomorrow. I cut each plug in half, placed it into a fresh eppendorf and incubated 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.

In preparation for PFGE and running my remaining PCR product tomorrow I checked over the equipment, ensured I had enough 1 x TAE buffer for standard gel electrophoresis and 0.5x TAE buffer for PFGE.

Tuesday 15th July 2014

Today started with collecting yesterday’s bacterial plugs from incubation, removing the buffer/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.

IMG_5085

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 de stain

In order to prepare the next batch of plugs for tomorrow I again cut each plug in half, placed it into a fresh eppendorf and incubated 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.

As I did not have time to run my last two batches of PCR product the week before last I then set up and ran the remaining product on 1.8% agarose gels for 1.5 hours, alongside 50 bp ladders for reference. I stained the gels for 15 minutes before viewing under UV. Similarly to my previous gels from the same PCR batch, the resulting gels were very disappointing, demonstrating clear and accurate banding for the 50bp ladder but not a single band for any of the samples. The negative control showed no banding and the positive control showed a clear band approximately 415bp in size which confirmed that my PCR had worked correctly.

In order to investigate this further I decided to re test the 1 in 1000 diluted samples I ran through PCR on the Nanodrop. As I expected the samples were far too diluted and were therefore unsuitable for use in PCR. I re diluted a selection of samples from both collections (this time 1 in 10), and then re tested these alongside the original undiluted samples on the Nanodrop. These samples were much better, demonstrating DNA concentrations once diluted of approximately 1-5 ng/μl – a lot more appropriate for PCR!

IMG_6838

I spent the rest of the day preparing to re run PCR tomorrow, gathering together PCR tubes, domed lids, Taq 2x mastermix, positive/negative controls (NCTC 13353 and ATCC 15922), x10 primers and diluting all my Lincoln and Bradford samples 1 in 10.

Wednesday 16th July 2014

As with yesterday today started with collecting yesterday’s bacterial plugs from incubation, removing the buffer/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.

Once my overnight PFGE had finished I removed the gel, staining it for 30 minutes and de staining for 10 minutes before viewing under UV. The resulting gel was as successful as my previous gels demonstrating accurate banding for the PFG and Lambda ladders as well as variable banding patterns for Bradford Escherichia coli samples.

My new PFGE 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 de stain

IMG_6840

In order to prepare the final batch of plugs for tomorrow I again cut each plug in half, placed it into a fresh eppendorf and incubated 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.

I decided to run a test PCR, before running all my samples again in order to confirm that my established PCR settings were definitely appropriate. As before I carefully pipetted the required reagents into 50μl PCR tubes, including mastermix Taq2x, all multiplex group primers (1 in 10 dilution), positive/negative controls (NCTC 13353 and ATCC 25922) and pure water. I then placed this into the PCR machine, and using the settings I used for my Lincoln/Bradford PCR, ran for 2 hours. Once completed 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 this gel for 15 minutes before viewing under UV. The resulting gel confirmed that my settings were appropriate, demonstrating clear and accurate banding for the 50bp ladder, no banding for the negative control and a clear band approximately 415bp in size for my positive control. I therefore concluded that my sample DNA concentration was the main problem for no visible bands on my previous PCR gels.

Thursday 17th July 2014

Again, today started with collecting yesterday’s final bacterial plugs from incubation, removing the buffer/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.

Once my overnight PFGE had finished I removed the gel, staining it for 30 minutes and de staining for 10 minutes before viewing under UV. The resulting gel was as successful as my previous gels demonstrating accurate banding for the PFG and Lambda ladders as well as variable banding patterns for Bradford Escherichia coli samples.

The new PFGE 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 de stain

I spent the rest of the day preparing all PCR reactions within PCR tubes. Again, I decided to run two separate PCRs, one for all Lincoln samples (Including a positive and negative control) and another for all Bradford samples (Again, including a positive and negative control). To ensure accuracy I prepared tables for each, which demonstrated the exact position of each sample within a 8 (A, B, C, D, E, F, G, H) by 12 grid. As before I used NCTC 13353 as my positive control and ATCC 25922 as my negative control.

I started with my Lincoln samples and carefully pipetted the required reagents into 50μl PCR tubes, including mastermix Taq2x, all multiplex group primers (1 in 10 dilution), plasmid DNA (1 in 10 dilution) and pure water. I then placed this into the PCR machine, and using the settings I have established from previous practice PCRs ran for 2 hours. I then repeated this process for the second PCR, using all my Bradford samples.

IMG_6442

Once both PCRs had finished it was quite late so I decided to store the PCR product overnight in the fridge before running the samples tomorrow.

Friday 18th July 2014

Once my final overnight PFGE had finished I removed the gel, staining it for 30 minutes and de staining for 10 minutes before viewing under UV. The resulting gel was as successful as my previous gels demonstrating accurate banding for the PFG and Lambda ladders as well as variable banding patterns for Bradford Escherichia coli samples.

I spent the rest of the day running the resulting PCR product for both the Lincoln and Bradford samples on 1.8% agarose gels for 1.5 hours, alongside 50 bp ladders for reference. I stained the gels for 15 minutes before viewing under UV. The resulting gels were very promising demonstrating clear and accurate banding for the 50bp ladder and bands for several of the samples. The negative control showed no banding and the positive control showed a clear band approximately 415bp in size which confirmed that my PCR had worked correctly. I managed to run and visualise gels 1, 2, 3 and 4 but ran out of time for gels 5 and 6 so they will have to be finished off tomorrow!

IMG_6849

Saturday 19th July 2014

I started today with the running of my final two gels. Again I ran the resulting PCR product on 1.8% agarose gels for 1.5 hours, alongside 50 bp ladders for reference. I stained the gels for 15 minutes before viewing under UV. The resulting gels were as good as yesterday’s gels, demonstrating clear and accurate banding for the 50bp ladder and bands for several of the samples. The negative control showed no banding and the positive control showed a clear band approximately 415bp in size which confirmed that my PCR had worked correctly.

I decided to re run those samples which demonstrated bands on my most recent PCR gels, to allow direct comparison between bands and the ladder. I used PCR product from Thursday 17th July and re ran the samples that demonstrated banding on 1.8% agarose gels for 1.5 hours, alongside 50 bp ladders for reference. I stained the gels for 15 minutes before viewing under UV. The resulting gels were as good as yesterday’s gels, demonstrating clear and accurate banding for the 50bp ladder and bands for every sample I used. The negative control showed no banding and the positive control showed a clear band approximately 415bp in size which confirmed that my PCR had worked correctly. This confirmed to me that these samples were definitely demonstrating bands and therefore indicated that these contained ESBL producing CTX-M Bla genes.

PCR CTX-M Multiplex Gel 1 19.7.14

As these samples were the final product from my PCR work for Lincoln and Bradford I decided to re run all the samples which demonstrated bands through PCR in order to improve clarity and double check the results. As on Thursday 17th July I carefully pipetted the required reagents into 50μl PCR tubes, including mastermix Taq2x, all multiplex group primers (1 in 10 dilution), plasmid DNA (1 in 10 dilution) and pure water. I then placed this into the PCR machine, and using the settings I have established from previous practice PCRs ran for 2 hours.

I spent the rest of the day running the resulting PCR product for both the Lincoln and Bradford samples on 1.8% agarose gels for 1.5 hours, alongside 50 bp ladders for reference. I stained the gels for 15 minutes before viewing under UV. The resulting gels were definitely worth repeating PCR, as although they demonstrated clear and accurate banding for the 50bp ladder and some bands which previously demonstrated bands no longer did. The negative control showed no banding and the positive control showed a clear band approximately 415bp in size which confirmed that my PCR had worked correctly. This allowed me to confirm that these samples were definitely demonstrating bands and that as a result of this these particular Escherichia coli contained ESBL producing CTX-M Bla genes.

PCR CTX-M Multiplex Gel 2 (2) 19.7.14

Next week…

Now I have officially finished my practical lab work I will be returning to Oxfordshire permanently to continue analysis of my PFGE gel images through Gel Compar as well as my PCR gel images from this week. I will now be working full time on completing my thesis, hopefully submitting a first draft to my supervisor at the beginning of September, when I am due to start my NEW JOB 😀

I plan on continuing to write a weekly update on my blog, however the focus will now be on the processes of analysis, writing up and submission rather than gathering data. Last year as part of the final year of my BSc I completed a research project which required me to write a dissertation. I also completed a write up for my UROS project which took place during the summer between my second and third year. Hopefully since I have some practise in writing up research project work (both in analytical chemistry and microbiology/molecular biology) writing my thesis won’t be too painful a process!!

A Year in the Lab…

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