Life After Lincoln – My New Blog!

When I begin writing this blog in early August 2013 I always intended that ‘A Year in the Life…’ would be just that, a year’s collection of blog posts that made up my experiences and life while studying at Lincoln University.

As a result, when I completed my research at Lincoln in August 2014 I decided to stop posting on ‘A Year in the Life…’ in order to focus on writing my thesis and begin to find my feet as a science graduate.

I am now spending a year gaining experience working in a secondary school ahead of applying to train as a science teacher, specifically biology. I was lucky enough to begin working as a teaching assistant back home in Oxfordshire, in September 2014 and have now been at my school for 5 months!

My time there so far has been amazingly good fun, really rewarding and has definitely confirmed to me that I want to teach. As a result I am now in the exciting stages of applying for teacher training!! I plan to use this new blog ‘Life After Lincoln’ as a way to record my experiences of life after university, both professionally and personally.

I hope that this new blog will be as interesting and informative to read as my previous and that you continue to follow me from new graduate to biology teacher 🙂

– Amy

https://lifeafterlincoln.wordpress.com

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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…

A Year in the Lab: Moving out of Lincoln and Running PCR on Lincoln and Bradford samples

Monday 30th June 2014

As I am moving out of my flat in Lincoln this week, research work had to take a bit of a back seat! I started the week still in Lincoln, preparing to run all plasmid positive samples through my established PCR. I selected the samples I wanted to process then calculated the amount of PCR tubes, positive/negative controls and reagents I will need. Once these were prepared I calculated the number of gels I will need to run in order to visualise all the samples. I then gathered together gel electrophoresis tanks and gel boxes, before ensuring I had enough 1x TAE buffer, loading buffer and 50bp ladder. I autoclaved my prepared buffer and chilled it in the fridge prior to use.

IMG_4819

Thursday 3rd July 2014

Today began with the preparation of all PCR reactions within PCR tubes. 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 1000 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 4th July 2014

Friday started off with a supervisor meeting in which we discussed both mine and other postgraduate’s research progression over the last month, as well as changes that will be occurring within the College of Science (Including our move to Joseph Banks Laboratory, which unfortunately I will just be missing!).

Once finished 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 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. As a result of this the lack of any sample bands either means that my sample DNA was too diluted (1 in 1000) or I don’t not have any samples which contain ESBL producing CTX-M Bla genes.

IMG_6849

Unfortunately I ran out of time to run and visualise my last two gels, or re perform PCR with adjusted settings so I decided to put research work on hold until I am back in a weeks time.

Next week…

As I have now officially moved out of my flat in Lincoln, I will be spending the next week back in Oxfordshire completing a few days of induction for my new job which starts in September 🙂 I will then be returning to Lincoln and staying at a friend’s house for a week in order to finish off my final research work before focusing entirely on writing up my thesis. The plan for my final week is to finish running my last 3 PFGE gels and input the resulting gel images into Gel Compar software before conducting comparative analysis during August. I will also be running and visualising my last two PCR gels before re performing with adjusted DNA concentration/dilution after Nanodrop testing.

A Year in the Lab…

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

Monday 23rd June

This week began with the production of my final bacterial plugs! As I have reached the end of both Lincoln and Bradford collections I started by inoculating LB broth with single colonies from my last agar slopes. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm.

During this 3 hour incubation I removed my overnight PFGE gel, stained it for 30 minutes and de stained for 10 minutes before viewing under UV. The resulting gel was very successful, demonstrating accurate banding for the PFG and Lambda ladders as well as variable banding patterns for Lincoln Escherichia coli samples. This is in line with what I expected to see – a variety of Escherichia coli isolates that have differing bacterial genomes that when restriction digested produce characteristic banding patterns on PFGE. Once all PFGE has been conducted these images can then be compared using the Gel Compar software I was trained on at the beginning of June!

Gel ComparGel compar Software (Biosystematica, 2013)

In preparation for running my next batch of plugs tomorrow I then 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.

Once the correct OD was reached for my new plugs, 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.

In order to set up my next PFGE gel I then collected 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

Finally I decided to prepare and run my next PCR, in order to test whether primer concentration was causing my fragment band (415bp) to appear thicker and darker when visualised under UV. I began by preparing a primer concentration gradient, ranging from x10 (Which I used in my PCR last week) down to x3, combining my x100 stock with pure filtered water to create each dilution. Again I just used my CTX-M group 1 primers and this was conducted for both the forward and reverse primer.

As before I used NCTC 13353 as my positive control and ATCC 25922 as my 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 used the most appropriate temperature I concluded from my last PCR – 62ºC, keeping the rest of my settings the same as before.

After 2 hours my PCR finished, however as it was fairly late in the day I decided to store the PCR product overnight in the fridge before running it tomorrow.

Tuesday 24th June

Today started by setting up and running my PCR product from yesterday 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 a little messier than previously, however it clearly and accurately demonstrated banding for both my 50bp ladder and samples. My negative control showed no banding, and my primer gradient demonstrated that primer concentration did not have a major effect on the darkness/thickness of the banding. As a result of this gel my next PCR will need to assess whether the concentration or purity of my extracted NCTC 13353 sample is an issue.

PCR Primer Cocnentration Gradient

I then continued by washing my final 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 they can be restriction digested.

Once my overnight PFGE gel had finished I stained it for 30 minutes and de stained for 10 minutes before viewing under UV. The resulting gel was as successful as yesterday’s demonstrating accurate banding for the PFG and Lambda ladders as well as variable banding patterns for Lincoln Escherichia coli samples.

In preparation for running my next batch of plugs tomorrow I then 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 order to set up my next PFGE gel I then collected 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.

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

Based on my PCR from yesterday I decided to measure the purity and concentration of DNA within my NCTC 13353 and ATCC 25922 control samples. As before I used the Nanodrop to test my extracted DNA samples in order to measure DNA in ng/μl, A260, A280, 260/280 and 260/230. 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. Both my samples demonstrated pretty good purity, however both were far too concentrated – NCTC 13353 at 1229.8 ng/μl and ATCC 25922 at 1373.2 ng/μl. When conducting a PCR, DNA concentration should generally be around 1 ng/μl, so naturally the concentration was way too high! I will therefore need to dilute my samples down by around 1000, to reach this appropriate concentration.

nanodrop2000spectrophotometerNanoDrop2000 from Thermoscientific (Bioresearch Online, 2013)

Wednesday 25th June

In preparation for running my next batch of plugs tomorrow I began the day by cutting each plug in half, placing it into a fresh eppendorf and 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.

Once my overnight PFGE gel had finished I stained it for 30 minutes and de stained for 10 minutes before viewing under UV. The resulting gel was as successful as yesterday’s demonstrating accurate banding for the PFG and Lambda ladders as well as variable banding patterns for Lincoln Escherichia coli samples.

In order to set up my next PFGE gel I then collected 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.

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

Finally I decided to continue my PCR work from yesterday, in order to test out the differences in DNA concentration before and after dilution. I began by diluting my NCTC 13353 DNA sample 1 in 1000, using pure filtered water to achieve the desired 1 ng/μl. Again I just used my CTX-M group 1 primers (diluted x10), based on my previous PCR. As before I used undiluted NCTC 13353 as my positive control and undiluted ATCC 25922 as my 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 used the most appropriate temperature I concluded from a previous PCR – 62ºC, keeping the rest of my settings the same as before.

After 2 hours my PCR finished, however as it was fairly late in the day I decided to store the PCR product overnight in the fridge before running it tomorrow.

Thursday 26th June

Today started by setting up and running my PCR product from yesterday 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.

IMG_6699

The resulting gel was very successful, demonstrating clear and accurate banding for the 50bp ladder and samples. My negative control showed no banding, while the diluted NCTC 13353 DNA sample showed a much clearer band than the undiluted NCTC 13353 DNA sample. This confirmed that diluting the NCTC 13353 DNA sample 1 in 1000 gave a more appropriate concentration of DNA for PCR.

In preparation for running my next batch of plugs tomorrow I then 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.

Once my overnight PFGE gel had finished I stained it for 30 minutes and de stained for 10 minutes before viewing under UV. The resulting gel was as successful as yesterday’s demonstrating accurate banding for the PFG and Lambda ladders as well as variable banding patterns for Lincoln and Bradford Escherichia coli samples.

In order to set up my next PFGE gel I then collected 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.

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

Friday 27th June

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

Unfortunately I ran out of restriction enzyme again, as my second delivery hasn’t arrived yet, so I couldn’t prepare any further Bradford plugs for PFGE. Instead I collected 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.

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

Finally I decided to confirm my established settings for PCR, by using my previously determined primer concentration (x10), diluted DNA (1 in 1000 – 1 ng/μl) and temperature (62ºC). Again I just used my CTX-M group 1 primers, as well as NCTC 13353 as my positive control and ATCC 25922 as my 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 kept the rest of my settings the same as before.

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 very good, demonstrating clear and accurate banding for the 50bp ladder and samples. My negative control showed no banding, and my two repeats confirmed that primer concentration (x10), diluted DNA (1 in 1000 – 1 ng/μl) and temperature gradient (62ºC) were appropriate for this PCR.

PCR Settings Confirmation

Saturday 28th June

As I couldn’t prepare any further bacterial plugs for PFGE yesterday, today just involved removing my overnight PFGE gel, staining it for 30 minutes and de staining for 10 minutes before viewing under UV. The resulting gel was as successful as yesterday’s demonstrating accurate banding for the PFG and Lambda ladders as well as variable banding patterns for Bradford Escherichia coli samples.

Next week…

As I am moving out of my flat in Lincoln next Monday my time in the lab will be limited to Monday, Thursday and Friday. Hopefully in this time my latest delivery of restriction enzyme will arrive and I can finish up restriction digesting and running Bradford bacterial plugs through PFGE. I can then begin comparing the individual sample profiles using the Gel Compar software. I will also be setting up, running and visulising my first attempt at PCR using my extracted Lincoln and Bradford collection DNA. Finally, I have a catch up research group meeting on Friday with my supervisors and other fellow postgraduate students!

A Year in the Lab…

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

Monday 16th June 2014

This week began by inoculating LB broth with single colonies from my Bradford 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.

IMG_5689

Tuesday 17th June 2014

Today started 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.

I then prepared and labelled LB broths ready for inoculation tomorrow morning.

Wednesday 18th June 2014

As on Monday today consisted of inoculating LB broth with single colonies from my Bradford 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.

Unfortunately the QIAcube is going to be out of action for a while until an engineer from QIAgen can come to have a look at it, so I decided it would be better for me to extract DNA from my PCR reference strains by hand, through alkaline lysis as I have done before. I therefore inoculated LB broths with single colonies from my previously produced streak plates and incubated these overnight, shaking at 37ºC.

IMG_6471

Thursday 19th June 2014

As on Tuesday today started 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 tomorrow (Now my restriction enzyme has arrived!).

I then collected my overnight reference strain LB broths from incubation to begin DNA extraction. This was conducted as before, starting by ensuring I had a waterbath set at 60ºC, labelled eppendorfs and 95% ethanol on ice, along with all other prepared reagents and chemicals. I began by pipetting 1ml of incubated sample into an eppendorf, centrifuging it for 5 minutes to form a pellet at the bottom of the eppendorf. I then removed the supernatant and re suspended the pellet in 100μl of TE buffer and 2μg of Lysozyme (100μl TE buffer + 2μl 50mg/ml Lysozyme Stock). This was then incubated for 30 minutes at 37ºC with agitation. 50μl of 10% SDS was added before incubating in a waterbath at 60ºC for 30 minutes. The sample was again centrifuged for 5 minutes, before remove the supernatant to a fresh eppendorf, discarding the old. 100μl of TE buffer was added and then, working in a fume cupboard, 250μl phenol chloroform isoamylalcohol was added. Ensuring the eppendorf is closed securely it was mixed for 1 minute (The mixture will appear white and cloudy). This was centrifuged for 5 minutes, and again in the fume cupboard, the top aqueous layer was carefully removed to a fresh eppendorf. I then added 650μl ice cold 95% ethanol, and incubated on ice overnight.

IMG_4989 Saturday 21st June 2014

I started today by inoculating LB broth with single colonies from my Bradford 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 then finished off my DNA extraction from yesterday, by centrifuging for 5 minutes. I removed the supernatant and re suspended the pellet in 70% ethanol. Again I centrifuged for 5 minutes. The supernatent was then removed and discarded, before air drying the eppendorf until all ethanol had evaporated off. Finally 30μl of distilled water was added and allowed to incubate at room temperature for 5 minutes.

In preparation for running my first batch of new plugs tomorrow I cut each the first 14 plugs from the Lincoln collection in half, placed them into a fresh eppendorf and 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.

Sunday 22nd June 2014

As before, today started 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 they can be restriction digested tomorrow.

I then collected 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 destain

I ran this gel using the same settings as my last most successful PFGE gel as confirmed last week.

In preparation for running my next batch of new plugs 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.

Based on my failed first attempt at PCR last week I decided to try again with my new positive control sample. Again I just used my CTX-M group 1 primers, however I changed my positive control to the reference strain DNA from NCTC 13353, using ATCC 25922 DNA for 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 49.6ºC to 62ºC in order to optimise the most suitable temperature for group 1 forward and reverse primers.

IMG_6703

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 much better than previously, demonstrating clear and accurate banding for both my 50bp ladder and samples. My negative control showed no banding, and my temperature gradient demonstrated that 62ºC was the most appropriate temperature out of those tested. I did notice however that my banding was very thick and dark. This could be as a result of using too concentrated primer and/or DNA.

PCR CTX-M Group 1 NCTC 13353 22.06.14

Next week…

I will be continuing to produce bacterial plugs from my Bradford samples, conducting restriction digests and then running these plugs, along with my previously produced Lincoln plugs, through PFGE to analyse strain. Based on my PCR work this week I plan on conducting both primer and DNA concentration gradients, as well as using the Nanodrop to analyse the purity and amount of DNA present within my extracted NCTC 13353 samples.

A Year in the Lab…