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…

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