A Year in the Lab: PFGE Plug Production and Antibiotic Resistance Testing

Monday 20th January 2014

I actually started the week on Sunday night at 6.30pm, inoculating 3 10ml nutrient broths with a single colony from streak plates of 3 identified isolates of Escherichia coli. At 7pm these were incubated at 37ºC with agitation, and then removed at 11am after 16 hours incubation in order to obtain optimum growth/number of bacterial cells.

In order to reacquaint myself with genotypic analysis before beginning work with PFGE I started the week by running through:

  • DNA extraction – Phenol chloroform isoamylalcohol
  • Restriction digestion – NotI
  • Gel electrophoresis – Standard 110V 1.5hr run

DNA extraction is performed on an incubated bacterial sample, in order to extract and purify DNA from within individual bacterial cell nuclei before conducting a restriction digest. I began by ensuring I had a waterbath set at 60ºC, labelled eppendorfs and 95% ethanol on ice, along with all other prepared reagents and chemicals from last week.

How to conduct DNA extraction

  • Pipette 1ml of incubated sample into a 1.5ml eppendorf, centrifuge for 2 minutes at 13,000 rpm – forming a pellet at the bottom of the eppendorf.

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  • Carefully remove the supernatant and resuspend the pellet in 100μl of TE buffer and 2μg of Lysozyme (100μl TE buffer + 2μl 50mg/ml Lysozyme Stock).
  • Incubate for 30 minutes at 37ºC with agitation.
  • Remove and add 50μl 10% SDS.

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  • Incubate in a waterbath at 60ºC for 30 minutes.
  • Remove and centrifuge for 5 minutes at 13,000 rpm.
  • Remove the supernatant to a fresh eppendorf, discarding the old.
  • Add 100μl TE buffer and then, working in a fume cupboard, add 250μl phenol chloroform isoamylalcohol.

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  • Ensure the eppendorf is closed securely and mix for 1 minute (Mixture will appear white and cloudy).

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  • Centrifuge for 5 minutes at 13,000 rpm.
  • Again in the fume cupboard, carefully remove the top aqueous layer to a fresh eppendorf then repeat the phenol extraction by adding 250μl phenol chloroform isoamylalcohol.

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  • Ensure the eppendorf is closed securely and mix for 1 minute.
  • Centrifuge for 5 minutes at 13,000 rpm.
  • Again in the fume cupboard, carefully remove the top aqueous layer to a fresh eppendorf then add 250μl of chloroform.

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  • Ensure the eppendorf is closed securely and mix for 1 minute.
  • Centrifuge for 5 minutes at 13,000 rpm.
  • Repeat chloroform extraction if sample appears cloudy.
  • If not, remove the aqueous layer to a fresh eppendorf then add 650μl ice cold 95% ethanol.

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  • Incubate overnight on ice.

During the first 30 minute incubation in lysozyme I inoculated nutrient agar slopes, which I prepared last week, with isolated colonies from Brayford water sample (B2). I did this by taking a single colony from a streaked plate on a disposable loop and gently spreading it from the lowest part of the slope upwards. I inoculated 2 agar slopes per isolated organism and left them on my bench to slowly grow single colonies, from which I can work from over time.

During the second 30 minute incubation in 10% SDS I made up some more nutrient broth for inoculating isolated organisms in on further water sample collection. I prepared 30 10ml broths as before, dissolving the powder in distilled water, decanting into glass universals before autoclaving.

When I had reached the overnight incubation on ice stage, I decided to practise using the antibiotic disc plunger on my Mueller Hinton agar plates. I have used the plunger before during my undergraduate degree, however it has been a long time and I have never set it up entirely by myself. I selected 8 antibiotics from the collection which are always stored in the labs for practical classes to try out:

  • RD – Rifampicin
  • CN – Gentamicin
  • TE – Tetracycline
  • S – Streptomycin
  • AMP – Ampicillin
  • P – Penicillin
  • PB – Polymyxin B
  • VA – Vancomycin

How to conduct antibiotic disc testing

  • Begin by setting up an aseptic workspace, ensuring that labelled Mueller Hinton agar plates, samples to be spread, plastic spreaders, appropriate pipette tips and pipette are within reach.
  • Remove the lid of chosen overnight culture, flame the neck and then remove 100μl of sample, flaming the neck again before replacing the lid.
  • Lifting the lid of your labelled Mueller Hinton agar plate, gently pipette the sample onto the surface of the plate, replacing the lid and disposing of the tip.
  • Take a fresh spreader, lift the plate lid and gently spread the sample evenly over the surface of the agar.

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  • Dispose of the spreader, replace the lid and leave the plate to dry for around 10 minutes.
  • Load the antibiotic disc plunger by gently pushing your chosen antibiotic tubes into the designated holes – listening for a click.

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  • Once dry, remove the lid of your chosen plate, place the plunger over the top of the plate so it is sitting on the bench.
  • Firmly and quickly push down on the plunger to release the discs onto the agar.

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  • Remove the plunger, check all discs are present then replace the plate lid.
  • Occasionally discs may not be released, if this happens remove the missing antibiotic disc tube from the plunger and set aside.
  • Dip forceps in ethanol and then flame to sterlise, before allowing to cool.
  • Remove 1 disc from the tube and carefully place onto the agar plate.
  • Ethanol and flame the forceps again before touching another disc or returning to the bench.
  • Incubate overnight at 37ºC.

Tuesday 21st January 2014

I began my day by finishing off DNA extraction:

  • Centrifuge for 5 minutes at 13,000 rpm.
  • Remove supernatant and resuspend the pellet in 70% ethanol.
  • Centrifuge for 5 minutes at 13,000 rpm.
  • Remove and discard the supernatant, then air dry the eppendorf until all ethanol has evaporated off.
  • Add 30μl of distilled water and allow to incubate at room temperature for 5 minutes.

I then continued by conducting a restriction digest on my extracted DNA, in order to ‘cut’ the DNA at specific points, creating fragments which can be seen and measured through gel electrophoresis. Different restriction enzymes can be chosen in order to cut DNA at specific places in the genetic code. For example, the enzyme I am using – NotI cuts DNA at GC/GGCCGC and was recommended for use in a protocol I am using to run identified Escherichia coli samples on PFGE.

How to conduct a restriction digestion

  • In a 1.5ml eppendorf, combine extracted DNA with chosen restriction enzyme, appropriate 10x restriction enzyme buffer and distilled water to make up to 30μl.
  • Mix gently to combine.
  • Incubate for an hour at 37ºC.

While waiting for the restriction digestion to incubate I observed the Mueller Hinton agar plates I incubated overnight. Using a ruler I measured the zones of inhibition visible around each antibiotic disc, noting down the result and photographing each plate. This measurement can then be compared against a reference standard supplied with the discs.

Generally speaking, the presence of clearing around certain antibiotics demonstrates the effectiveness of the antibiotic against the bacteria present on the plate. If a bacterial isolate is susceptible to an antibiotic the zone of inhibition will be larger due to it’s ability to inhibit growth of bacteria. Conversely if the bacteria demonstrates resistance to an antibiotic the zone of inhibition will be smaller/not present due to the inability of the antibiotic to prevent growth of the bacteria.

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I did notice that my first batch of Mueller Hinton agar plates were too thick, so I decided to make a second batch paying special attention to the thickness of each plate as I poured aseptically.

Once restriction digest incubation had finished, I began gel electrophoresis in order to separate DNA fragments present by individual size and charge. As a result, the smaller and more negatively charged molecules move faster, while the larger and less negatively charged molecules move slower through the gel. Therefore separation occurs due to individual electrophoretic mobility, and bands produced can be compared to known sized ladders.

How to prepare a 0.8% agarose gel

  • Begin by using masking tape to seal a gel casting box, making sure there is a tight seal between tape and box.
  • Place on a flat surface away from disturbances and position the comb at one end of the gel – ensuring it doesn’t touch the bottom of the tray.
  • Prepare a 0.8% agarose gel by combining 0.8g of agarose powder with 100ml of 1x TAE buffer in a conical flask, topping with grease proof paper or a large lid to prevent fluid loss through evaporation.

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  • Heat in a microwave for around 2 minutes, using short bursts to ensure boiling and gently swirling to combine.
  • Once gel appears clear and homogeneous remove carefully and leave to cool on bench.

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  • When you can comfortably hold the base of the flask in your hand, carefully pour the cooled agarose into the taped tray, ensuring no bubbles are formed.

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  • While the solution is still liquid you can use a clean pipette tip to pop/drag any bubbles to the sides, but be careful not to do this once setting has begun.
  • Allow to set for at least 15 minutes.

How to load and run gel electrophoresis

  • Begin by gently removing the comb from the set agarose gel, removing tape around gel box and placing it into an electrophoresis tank.

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  • Ensure that your wells are nearest the negative electrode or black lead, as DNA is negatively charged and will move towards the positive electrode or red lead.
  • Produce 1L of 1x TAE buffer by combining 20ml of 50x TAE buffer stock with 980ml of distilled water in a volumetric flask and gently combine.

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  • Fill both ends of the electrophoresis tank, allowing the buffer to cover the agarose gel by around 3mm, but not reach the overfill line.
  • Prepare loading solutions in 1.5ml eppendorfs before carefully pipetting 20μl of each sample into a separate well, including a loading buffer to allow visualisation of DNA fragment progression (This does take a bit of practise as you need a steady hand and to release the solution just above the well, removing the pipette from the buffer before releasing entirely).

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  • Pipette your chosen appropriate ladder to allow comparison and calculation of fragement/band size.
  • Ensure the power pack is switched off, place the lid onto the electrophoresis tank.

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  • Attach the leads to the power pack, set to your requirements (In my case 110V for 1.5 hours) and confirm.
  • Check that bubbles are forming at both ends of the tank then leave to run.

How to stain and view the gel

  • Once finished, switch off the power pack and remove the lid.
  • Wearing gloves, gently lift the gel box out of the tank and slide the gel off into a large plastic tray.
  • Tip the TAE buffer into the tray and add 5μl Ethidium Bromide to the buffer (As Ethidium Bromide is carcinogenic ensure that you are wearing gloves and dispose of used equipment appropriately).

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  • Allow to stain for 30 minutes before visulising using an imaging system – in my case Kodak Gel Logic 100.
  • When finished dispose of your gel into the same carcinogenic waste as before.

Once finished, gel observed and the image saved I reviewed and prepared PFGE reagents and chemicals for use tomorrow. I decided to repeat antibiotic testing using my new thinner Mueller Hinton agar plates, following the same procedure as yesterday – incubating overnight at 37ºC.

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In preparation for plugs production tomorrow I inoculated 3 10ml nutrient broths with an individual single colony of 3 identified isolates of Escherichia coli. At 7pm these incubated at 37ºC with agitation, and were removed at 11am after 16 hours incubation in order to obtain optimum growth/number of bacterial cells.

Wednesday 22nd January 2014

I started the day by removing my overnight nutrient broth sample and beginning PFGE plug preparation.

How to produce PFGE bacterial plugs

  • Add chloramphenicol and incubate for an hour at 37ºC with agitation.

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  • Melt the 2% agarose for a few seconds in the microwave before equilibrating the solution at 50ºC in a waterbath.
  • Centrifuge all 10ml of each sample in separate eppendorfs, in 1.5ml batches for 3 minutes at 130,000 rpm, removing and discarding the supernatant each time.
  • Resuspend the pellet in cell suspension buffer and equilibrate at 50ºC in a waterbath (I decided to make 1ml of 1% plugs and therefore the amount of cell suspension buffer and 2% agarose were the same – 500μl).

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  • Combine 2% agarose with cell suspension and mix gently.
  • Carefully pipette into plastic molds (85μl) and allow to solidify before moving to 4ºC for 10-15 minutes to add strength to the plugs before removal.

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While samples were undergoing chloramphenicol incubation I observed my antibiotic resistance testing plates, measuring zones of inhibition, making notes and taking photographs.

During plug pipetting it became very clear that the cell concentration within the plugs was too high, and even though plugs were left to set for an hour the integrity was too fragile to continue. I decided to try a lower initial amount of sample and 3 more 10ml nutrient broths were inoculated with an individual single colony of 3 identified isolates of Escherichia coli. Again these were incubated at 37ºC with agitation for 16 hours.

Thursday 23rd January 2014

Again, I began my day by removing my overnight nutrient broth sample and beginning PFGE plug preparation. I conducted the production in the same way as previously, however I decided to just use 2ml of sample. As I reached the pipetting stage the plugs felt a lot more stable and on removing them into individual eppendorfs they retained their integrity very well. I therefore continued the process:

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  • Remove each plug into an individual eppendorf.
  • Add lysozyme to each plug and gently mix.
  • Incubate for 2 hours at 37ºC.
  • Remove each plug gently from lysozyme, rinse with distilled water and replace into individual eppendorfs.

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  • Add proteinase K to each plug and gently mix.
  • Incubate overnight at 50ºC.

Once finished I decided to conduct some more antibiotic resistant testing, using 3 identified Escherichia coli samples from the bacterial isolate collection. I used the same procedure as previously, including the same antibiotics and decided to conduct repeats of each. Incubating overnight at 37ºC.

Friday 24th January 2014

I had a supervisor meeting from 10am-12pm, however as I needed to conduct the next washing stage of my plug production I began work at just before 10am. I started by preparing 1x wash buffer from a 10x wash buffer stock.

PFGE plug production: Washing Stage

  • Wash each plug in 1ml of 1x wash buffer for an hour each, with agitation at room temperature.
  • Repeat 4 times.
  • Store in 1x wash buffer at 4ºC.

Once completed I observed my overnight antibiotic resistance plates, recorded the zones of inhibition present and photographed each plate.

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Next week…

Now I have a first batch of plugs prepared I can conduct a restriction digest on the plugs on Monday, incubating overnight before running on Tuesday. Once the results from these plugs have been visualised I can either adjust my procedure to improve them or begin repeating the procedure on other identified Escherichia coli samples from the bacterial isolate collection. I am very happy with the antibiotic resistance testing procedure I have conducted this week, so I will continue researching, ordering and preparing reagents and chemicals for IEF next week.

After my supervisor meeting on Friday some aspects of my project have changed slightly so I will also be adjusting my research plans for the coming months and filling in a GS4 next week. Hopefully beginning my first large scale on site sample collection in the first week of February 2014!

A Year in the Lab…

A Year in the Lab: Genotypic Analysis and Gel Electrophoresis

After spending the last few weeks with family back home in Oxfordshire, and it is good to be back in Lincoln ready to start back in the lab. Now I have phenotypically identified all the organisms within the bacterial collection, I am moving onto working to genotypically analyse the organisms.

I am planning on using two specific techniques:

  • Agarose gel electrophoresis, specifically pulsed field gel electrophoresis (PFGE)
  • Isoeletric focusing (IEF)

I have worked with agarose gel electrophoresis and PFGE before, however IEF is a new technique to me. Standard electrophoresis works on the principle that one electrical field in one direction causes the separation of DNA and RNA, based on the sample’s size and charge. Therefore smaller and more negatively charged molecules move faster, while larger and less negatively charged molecules move slower through the gel. As a result separation occurs due to individual electrophoretic mobility, and bands produced can be compared to known sized ladders.

PFGE gel exampleOn the left – PFGE gel demonstrating bands created by DNA fragments moving through gel based on size and charge. On the right – Appropriately sized ladder, which is used as a comparison and allows calculation of unknown band fragment size.

In comparison, PFGE uses several electrical fields from multiple directions at interchangeable switch intervals. DNA or RNA present within a sample form a standard state, which when applied with electrical fields elongates and begins to travel through the gel according to charge and size.

PFGE microscopic DNA diagramTime lapsed microscopic images of DNA in sample during PFGE, showing standard state, elongation and travel through gel (Birren and Lai 1995)  

This change of direction and electrical field allows DNA that would previously have been too large, able to be separated. This is because DNA within the samples actually moves in a zigzag manner, due to the continuous change in electrical field direction, decreasing the length of agarose gel required for effective separation. As a result of this, PFGE is particularly useful for my research as it allows the separation of DNA over 10Mb/10,000Kb, separation of entire genomes and therefore analysis of individual bacterial strains.

CHEF electrical field diagramDiagram of CHEF-DR II PFGE electrical fields, acting on the DNA within a sample from multiple directions at interchangeable switch intervals, causing DNA to move in zigzag pattern through gel

PFGEPFGE equipment, including: CHEF-DR II unit, cooling system, buffer pump and power pack 

IEF allows the separation of soluble proteins within a sample based on charge, which I will use to investigate bacterial enzyme production.

I also hope to develop an antibiotic resistance measurement protocol from techniques I have used before. Starting by collecting a water sample from the Brayford, running through established sample preparation and bacterial isolation. Then incoulating mueller hinton agar and conducting antibiotic resistance testing with antibiotic discs.

Monday 13th January 2014

I began the week by preparing stocks and plates. As I want to conduct antibiotic resistance testing on isolated organisms from the Brayford using antibiotic discs I will need Mueller Hinton agar plates. I produced 20 plates of MacConkey, Nutrient and Mueller Hinton agar by combining appropriate agar powder with distilled water in a conical flask, gently mixing, sealing with cotton wool and grease proof paper before autoclaving. Once finished I allowed the agar to cool before pouring aspectically and allowing to set.

As I will be running PFGE, I decided to start by reacquainting myself with the technique by practising running a standard electrophoresis gel. For this I require TAE buffer, so I produced a 1L 50x TAE stock that can be diluted to use during both standard electrophoresis and PFGE.

How to produce 1L 50x TAE buffer stock

  • Begin by preparing an ethylenediaminetetraacetic acid (EDTA) solution.
  • For a 500ml 0.5M stock solution, add 93.05g of EDTA disodium salt powder to 400ml of distilled water and allow to dissolve on a magnetic hotplate with magnetic stirrer.

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  • The EDTA disodium salt powder won’t dissolve entirely until it reaches pH 8, therefore sodium hydroxide (NaOH) must be added to adjust the pH.
  • A 100ml 0.5M stock solution of NaOH was made by combining 2g of powder in 100ml of distilled water. The pH was measured using a pH probe and NaOH added until the pH reached 8 and all EDTA disodium salt powder dissolved.

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  • The solution was then topped up to 500ml using distilled water.
  • 242g of Tris-Base was then weighed out and dissolved in 750ml of distilled water.
  • 57.1ml of Glacial acetic acid was then added, followed by 100ml 0f the prepared 0.5M EDTA solution.

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  • Once completely combined, the solution was made up to 1L in a volumetric flask with distilled water.
  • This solution was then separated into two brown Winchester bottles (50ml each) and autoclaved.

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One finished I spent some time checking over and re setting up the PFGE equipment, as it was cleaned and packed away once we finished using it during the UROS project. I also did the same for the spiral plater and plate reader due to both being moved for lab fumigation.

Tuesday 14th January 2014

In order to conduct antibiotic resistance testing and establish a protocol I started the day by collecting a fresh water sample from the Brayford, using the same method as previously. On returning to the lab I inoculated nutrient broths with 50μl, 100μl, 500μl and 1000μl of water sample. Incubating these overnight at 37ºC.

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I spend the rest of the day writing and preparing a restriction digestion and gel electrophoresis procedure, based on previous work and research papers. Once finished I collected together and prepared any equipment, reagents and chemicals required. Including loading buffer diluted with 50% glycerol.

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Wednesday 15th January 2014

I began the day by observing the overnight nutrient broths, as expected universals were progressively cloudier from 50μl, 100μl, 500μl through to 1000μl. I then inoculated each sample onto MacConkey agar using the spiral plater and incubated overnight at 37ºC.

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I spent the rest of the day reviewing the DNA extraction, restriction digest and plug production for PFGE, as well as calculating reagent and chemical stocks.

Thursday 16th January 2014

I observed the incubated spiral plated MacConkey plates, isolating single colonies and inoculating each into separate nutrient broths. These were then incubated overnight at 37ºC. The isolated colonies were preliminary identified as:

  • Lactose positive violet – Enterobacter spp., Citrobacter spp. or Escherichia coli

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  • Lactose negative pink/cream – Proteus spp. or Pseudomonas spp.

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  • Lactose negative brown – Salmonella spp.IMG_4896
  • Lactose negative light pink – Serratia spp.

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Friday 17th January 2014

Overnight isolated colonies in nutrient broth were observed and streak plated onto nutrient agar, then stored on the lab bench over the weekend to allow slow growth ready for Monday. In order to work continuously with isolated organisms agar slopes can be used to allow storage for an extended period of time. I decided to produce 30 nutrient agar slopes.

How to make nutrient agar slopes

  • Begin by combining nutrient agar powder with distilled water in a conical flask (The amount you use will depend on how many slopes you want to make).

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  • Dissolve and heat on a magnetic hotplate with magnetic stirrer.
  • Once the mixture reaches boiling point, remove and allow to cool slightly before pouring 10ml into each glass universal.
  • When all universals are filled loosen the lids and autoclave.

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  • Once finished remove and place into a metal basket, leaning the basket at an angle to create a slope once set.
  • Leave slopes to set.

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I then spent the rest of the day writing and preparing a DNA extraction procedure, based on previous work and research papers. Once finished I collected together and prepared any equipment, reagents and chemicals required. Including: Tris-EDTA (TE) buffer, lysozyme, SDS and ethanol.

How to make TE buffer

  • Begin by producing a 1M 500ml stock of Tris-Cl, by adding 60.57g of tris in 500ml of distilled water, allowing to dissolve on a magnetic hotplate with magnetic stirrer.
  • Tris powder won’t dissolve entirely until it reaches pH 8, therefore concentrated hydrochloric acid (HCL) must be added to adjust the pH 8.
  • As I already had a stock of 0.5M EDTA prepared, I then combined 2ml of this with 10ml of prepared 1M Tris-Cl and 998ml of distilled water in a 1L volumetric flask.

How to make 50mg/ml lysozyme

  • Combine 0.5g of lysozyme with 5ml of TE buffer.
  • Gently mix to combine.

How to make 10% SDS

  • Combine 10g of SDS with 80ml of distilled water.
  • Gently mix to combine (SDS is a detergent and will create a lot of bubbles when mixed – so leave for a few minutes to settle before use).
  • Make up to 100ml with distilled water.

How to make 95% and 70% ethanol

  • Combine 9.5ml of 100% ethanol with 0.5ml distilled water.
  • Gently mix to combine.
  • Combine 7ml of 100% ethanol with 3ml distilled water.
  • Gently mix to combine.

Next week…

As I expected this week was mostly stock and reagent preparation, as well as procedure writing. I have four organisms isolated and streak plated ready to be inoculated on nutrient agar slopes next week. As well as mueller hinton agar ready to be inoculated and antibiotic resistance tested. I also have all reagents and equipment prepared to run a refresher standard gel using identified Escherichia coli samples from the bacterial collection. I hope that next week I will also be able to use these identified Escherichia coli samples to start producing plugs for use with PFGE.

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References

Birren and Lai, E. (1995) Pulsed field gel electrophoresis – A practical guide, Academic Press Inc., CA, USA.

A Year in the Lab…