Tag: PFGE

Hello Everyone Yesterday we ran a 24 hour…

Amy Thompson / / Uncategorized

Hello Everyone!

Yesterday we ran a 24 hour gel, at 4 V/cm with a 20 second switch interval PFGE gel. We used 3 large restriction enzyme treated plugs and a NEB ladder. Mike also asked us to research the genome for Propionibacterium acnes and the size of the fragments produced when lysed with SpeI. He asked us to do this so that we would be able to sketch a rough drawing of what we would really like to see on a perfect gel. Also to help us look for a more appropriate ladder to use with the Propionibacterium acnes samples, as the average size of a fragment is approx. 100kb, and the NEB yeast we are currently using runs from approx. 1900-225, not quite low enough to compare most fragments.

Today we observed the gel with Mike, which was how he expected it to look, with the NEB bands close together near the top of the gel. However, no bands were visible under the Propionibacterium acnes samples and Mike thinks we really need to have another go at making the plugs, perfecting the cell density and process. We also filmed our latest videoblog!

See you all on Monday 🙂

Hello Everyone Came into the lab today removed…

Amy Thompson / / Uncategorized

Hello Everyone!

Came into the lab today, removed, stained and viewed our latest gel. We observed a clear similar band under all the samples not treated with restriction enzyme, and slightly blurry similar band under all samples treated with restriction enzyme. Although the position of the bands are correct, the blurriness suggests that separation is still not occurring properly. There also appears to be a problem with the sealing agarose, resulting in very dark wells as DNA cannot properly leave the wells. This was actually my fault, as when making up the sealing agarose for the wells, i had been mixing the same 1g of agarose used in making the actual gel, in the smaller amount of buffer (50ml) used when making the sealing agarose, instead of halving the agarose to 0.5g. Therefore we were attempting to seal the wells with 2% agarose, which explains the difficulty we had in pipetting it (the formation of bubbles), the dark wells and smearing of samples through the gel.

We decided on the following modifications:

  • Increase the agarose gel % from 1% to 1.3/1.4%, in order to help increase the separation of fragments.
  • Use the correct agarose gel % to seal the plugs into the wells, hopefully decreasing the dark wells and smearing, and increasing the movement of DNA through the gel clearly.

We then ran a standard 0.8% agarose gel, with 0.5X TAE buffer, for 2 hours at 100V. We ran 3 large samples of restriction enzyme treated plugs, 1 Kb ladder and lambda Hind III ladder. We sealed the plugs in with 0.8% agarose (0.4g agarose and 50ml 0.5X TAE buffer).

The results of this confirmed our modifications, and tomorrow we will be running a new PFGE gel for 24 hours, at 4 V/cm with a 20 second switch interval, applying our modifications.

See you all tomorrow 🙂

Hey All Today we came in removed stained…

Amy Thompson / / Uncategorized

Hey All,

Today we came in removed, stained and viewed our gel. The samples not treated with restriction enzyme showed a similar band, just underneath the wells. This was expected as the samples had not been treated and therefore lysed by the restriction enzymes, so separation according to size did not occur, producing only one/two bands. The samples treated with restriction enzyme showed some separation, but not enough, possibly due to not enough digestion. For both sample types it appeared that the largest plug size used worked the best. Mike also noted that this may be due to a lack of cell density within the plugs. The agarose gel also appeared very messy, and needed to be cleaned up. We destained the gel in TAE buffer, viewed it again and decided on the following modifications for our next PFGE run:

  • Run for 20 hours with a 20 second switch interval. The switch interval was decreased in order to produce better/clearer separation.
  • 3 large samples for each plug treated with restriction enzyme and those without, as the larger samples seemed to work the best.
  • We also deep cleaned our glass wear and autoclaved the buffer used within the sealing agarose, in order to try to clean up our gel as best as possible.
  • Finally, when we produce more plugs, we will try to increase the cell density within the plugs and modify our cell suspension buffer/centrifuge stage.
  • We are keeping the buffer, ladder, sealing in plugs, temperature and voltage the same.

Excited to see if our modifications produce clearer band separation 🙂

Afternoon Everyone Started off today by viewing our…

Amy Thompson / / Uncategorized

Afternoon Everyone!

Started off today by viewing our latest gel with Mike. Unfortunately our samples ran completely off the gel, and therefore we only saw the areas where the samples had passed. We also saw that the wells were very dark, due to too much DNA still being present within them. We also used the wrong ladder for Propionibacterium acnes. Mike therefore decided that we should run our next gel for 24 hours, at 4V/cm with a 30 second switch interval time, based on our two best previous gels (PFGE 5 and 6). We also decided to cut our plugs into different sizes in order to see how the size of the plug would effect the gel. We cut the plugs into two small pieces, one medium piece and one large piece. We still used samples of both plugs treated with restriction enzyme and without. We also used a reference ladder of NEB yeast.

We then helped Mike to re culture the Propionibacterium acnes by making up TGYE agar, autoclaving it and pouring 15 plates 20ml each. Which i have seen done before, but never done myself, so it was good to have the opportunity to practise! Managed to pour them all pretty well 🙂

We came in at 5pm, to cut off the first well of the Propionibacterium acnes sample treated with restriction enzyme, to see it’s progress. We removed it, stained it and viewed it. We will interpret it when we have the final gel tomorrow.

See you all tomorrow, when our next gel comes out at 11am 🙂

Evening Everyone Didn’t come into the lab till…

Amy Thompson / / Uncategorized

Evening Everyone!

Didn’t come into the lab till the afternoon today. Once we got in we removed the buffer from the plugs, and incubated them further in 1X wash buffer for 30 mins, at room temperature. While this was incubating myself and Jo made up 3L of 0.5X TAE buffer, melted agarose and then poured a new gel. The buffer was then removed and replaced with some of the 0.5X TAE buffer, that will be used to run the gel, to allow equilibration. We then cut and loaded 3 samples of fully treated Propionibacterium acnes DNA, 3 samples of Propionibacterium acnes DNA not treated with the restriction enzyme (SpeI) and 1 sample of lambda Hind III as a reference ladder.

Finally we sealed the plugs in with agarose and set the PFGE to run for: 12.7 hours at 6.0 V/cm with a ramping of 1-8 seconds, then 7 hours at 6.0 V/cm with a ramping of 0.1-2 seconds. We haven’t tried ramping before, but we decided to try this set up, as it is the method used in one of the journals we have been working from. Ramping is where one switch interval is used at the beginning of a time run, and another is used at the end, therefore over the time period the ramping slowly changes.

Tomorrow we will be filming our next videoblog, removing, staining and viewing our gel. As mike won’t be in, we will be discussing modifications and a plan for next week on Monday.

See you all tomorrow! 🙂

Afternoon all Just to let you know the…

Michael Shaw / / Uncategorized

Afternoon all…Just to let you know the hard work that Amy and Jo have been doing to VLOG (Video blog) as well as blog their progress is now live on Youtube. Both this blog and the vlog are new experiments for Nicki and myself to see just how useful/effective different means of web-sharing can be; maybe it’ll become something we embed into teaching from hereon in, maybe it won’t…only one way to find out.

UROS VLOG on Youtube

Afternoon Everyone Pretty short day today Myself and…

Amy Thompson / / Uncategorized

Afternoon Everyone!

Pretty short day today, Myself and Jo came in at 11am to film our latest videoblog (I’m starting to really enjoy making them!) and talk through our previously filmed material from last week with Mike. Our footage won’t be up for you all to view for a little while yet, but I will let you know when Mike makes it available!

We then removed our latest gel from the PFGE, stained it and viewed it. We have made some initial observations; that the separations unfortunately ran off the end of the gel and we also sized up the bands using the information within the NEB yeast package.

Myself and Jo decided on some modifications between us, and we will be reviewing these with Mike on Monday morning.

Can’t believe week 2 of the project is over already! See you all next week! 🙂

Hello Everyone Myself and Jo got into the…

Amy Thompson / / Uncategorized

Hello Everyone!

Myself and Jo got into the lab early this morning, in order to set up our next PFGE run with our new modifications. We made up 3L of 0.5X TAE buffer and 150ml of agarose (in order to reduce error), then left the gel to set. While it was setting we made up 50ml of agarose to seal the plugs into the wells. We then cut up 4 samples of NEB yeast, pushed them into the wells and sealed them up with agarose. We then set up the PFGE equipment to run at 6°C, for 15 hours at 6 V/cm with a 70 second switch interval and then for 11 hours at 6 V/cm with a 120 second switch interval.

The run is to finish at 12.15pm tomorrow, so myself and jo will be in the lab from 11am to film our second videoblog section! 🙂

See you all tomorrow morning!

Evening all Myself and Jo spent this morning…

Amy Thompson / / Uncategorized

Evening all!

Myself and Jo spent this morning researching the next stages of our project, then we came into the lab for 3pm, in order to take our gel out of the PFGE. We then stained it up and viewed it with Mike. We saw much better separation than before, with the bands reaching all the way to the bottom of the gel, and running off. Both yeast samples, BioRad and NEB, also appeared consistent with each other. The 1Kb ladder was not visible, which was expected. However, the separations slightly shifted to the right, producing wonky bands, that unfortunately weren’t very defined. Mike said this was most likely due to imperfections in the agarose, and we could rectify this by producing 150ml of agarose in order to reduce error. In order to try and stop the yeast sample running off the gel we decided to change our switch interval as well. We also decided that as the NEB yeast has been the most consistent and successful sample, we would stop running the BioRad yeast and 1Kb ladder. We will keep the size of the plugs the same, and well as sealing them with agarose.

Reviewing the NEB yeast information we decided to try running our next gel following the guidelines provided, 1% agarose, 15 hours at 6 V/cm with a 70 second switch interval and then 11 hours at 6 V/cm with a 120 second switch interval. Using 0.5X TAE buffer, 2 samples of NEB yeast and at 6°C as before.

See you all tomorrow morning as we set up our next, and possible last gel before starting on our next stage in the project! 🙂

Afternoon Everyone Myself and Jo came in at…

Amy Thompson / / Uncategorized

Afternoon Everyone!

Myself and Jo came in at 9am this morning, and began filling out our COSHH and risk assessment forms. Although they were a bit of a challenge we managed to get them finished before our PFGE gel came out at 11.30am. Filling out the COSHH form involved making a list of all the chemicals we have used so far within our project, looking up their hazard category (using MDSS – which was very useful!), assessing their exposure potential (glacial acetic acid and ethidium bromide being our highest), steps we could take to protect ourselves (containment level), first aid and how to dispose of them. Our containment level for procedure was 1. The Risk assessment form involved identifying possible hazards within the lab, reviewing control measures we could take to reduce this risk and giving an overall risk assessment rating, ours was medium. I felt that filling out these forms was good practise as next year, during our final year projects, we will be required to do this ourselves for our own projects.

We also watched Mike re culture the Propionibacterium acnes in the anaerobic cabinet, which was really interesting to see!

We then removed, stained and viewed our gel. We managed to obtain much clearer bands and we no longer had very dark areas (large amounts of DNA) within the wells. However we did notice slight band elongation, which is caused by increased temperature. This is expected, as unfortunately the PFGE is still running at 25/26°C, instead of the 14°C it really should be running at.

We therefore decided to keep the smaller amount of DNA we were putting in the wells, keep sealing in the plugs, keep the buffer at 0.5X and not include the initial switch interval as before. In order to reduce the temperature and obtain clearer bands we decided to move the PFGE equipment into the fridge (to run at 6°C). In order to obtain more separation we decided to increase the voltage to 6.0 V/cm. We therefore set up and started a new run with these changes for 24 hours at 6.0 V/cm and with a 45 second switch interval, still using 2 samples of NEB yeast, 2 samples of BioRad yeast and 2 samples of 1Kb ladder. With a second block for 2 hours at 0.6 V/cm and with a 45 second switch interval, in case we weren’t ready to remove the gel.

Really excited to see how the massive change in temperature effects our separations! 🙂

See you all tomorrow!

Afternoon All Quite a quick day today Myself…

Amy Thompson / / Uncategorized

Afternoon All,

Quite a quick day today! Myself and Mike viewed the PFGE gel we ran last Thursday to Friday. Although the run appeared better in terms of visible band separations, the bands were still fairly faint and the wells appeared very dark. Mike said that this was due to too much DNA being left in the wells, we therefore decided to reduce the amount of plug we put into the well, by cutting the plugs as small as we could. We decided to reduce the buffer %, from 1% to 0.5%, to get rid of the initial 3 hour with 15 second switch time at 2.0 V/cm and add a second band marker (1Kb ladder). This 1Kb ladder was added as a reference in order to help us work out how many bases were present within samples.

We set up a PFGE run with all these new changes for 24 hours at 2.0 V/cm with a 45 second switch interval, and a second block for 1 hour at 0.6 V/cm with a 45 second switch interval in case we weren’t ready tomorrow morning to remove the gel. So we will see tomorrow what our changes have done! 🙂

Mike also showed me the TGYE agar he was making up, in order to re culture the Propionibacterium acnes samples. He showed me how to make up the agar, autoclave it and pour it. Tomorrow he is going to show me how to streak plate the Propionibacterium acnes samples onto the TGYE agar plates in the anaerobic cabinet. I have never used it before, so it should be really good to see!

See you all tomorrow! 🙂

Hey Everyone Can’t believe I have finished my…

Amy Thompson / / Uncategorized

Hey Everyone!

Can’t believe I have finished my first week in the labs already! Myself and Jo came in at 10am this morning and filmed our first video blog 🙂 It went pretty well! 🙂

We filmed an intro about who we are, what we are doing, where we are doing and why we are doing it, as well as how we are doing it, through a summery of our first week in the lab – our achievements, problems, solutions and results.

When we had finished we removed our gel from the PFGE and stained it in Ethidium Bromide, while staining we emptied the PFGE of buffer. After staining we viewed the gel under UV, as Mike wasn’t in the lab today, we saved the image for Monday.

Can’t wait for next week! See you all soon 🙂

Hey All Didn’t do very much today as…

Amy Thompson / / Uncategorized

Hey All! 🙂

Didn’t do very much today, as we are trying a 24 hour run with our new modifications we decided on yesterday. Jo and I came in at 9am, and made up 3L of TAE buffer, and left it to chill while we prepared our agarose gel. After we poured it and left it to set (which took a few goes today, as the gel box wasn’t tightened enough and gel kept escaping!) we prepared our samples. We used 2 repeats of BioRad yeast, 2 repeats of NEB yeast and loading buffer as a reference. For this run we also decided to seal the sample plugs within the wells with agarose, in order to promote the movement of the DNA from the wells and into the gel. We used a pipette tip cut with a knife, to give a larger, slanted tip, from which we pipetted agarose into the wells. This worked very well, and we managed to fill the wells easily. We then placed the gel into the PFGE, added buffer and our loading buffer. We turned on the pump to expel any bubbles trapped within the pump/tubes, and then ran the PFGE for 3 hours at 2.0 V/cm with a field switching time of 15 seconds. We decided to do this initial stage to give the DNA time to start moving into the gel, and hopefully allow clearer band separations.

At 2pm we returned to the lab to reprogramm the PFGE to run for 21 hours at 2.0 V/cm with a field switching time of 45 seconds, followed by 2 hours at 0.6 V/cm with a field switching time of 45 seconds. This run should finish at 11am tomorrow morning, therefore the second programm was put in incase we were not quite ready to remove the gel.

Tomorrow morning we will hopefully get to see how our modifications have improved our separations, and if not discuss further modifications for next week. We will also be filming our first video blog!

See you all tomorrow! 🙂

Evening Everyone As soon as Me and Jo…

Amy Thompson / / Uncategorized

Evening Everyone,

As soon as Me and Jo got into the lab today, we made up 3L of TAE buffer, and left it to chill until we needed it. We then turned off the PFGE, as it had been running overnight, removed the gel and placed it into Ethidium Bromide for 30 minutes to stain. While it was staining we emptied the PFGE of buffer. We then analysed the gel with Mike under UV.
The separations appeared blurry and unclear, Mike told us that this was most likely due to contamination with nucleases, and too low field switching (the time it takes for the electric fields to switch from 1 to 2). We therefore decided to increase our field swtiching time from 1 second to 45 seconds (using ‘Pulsed Field Gel Electrophoresis – A practical guide’ Birren, B. and Lai, E). We prepared another gel, with 10 lanes, containing 3 repeats of BioRad yeast, 3 repeats of NEB yeast, 3 repeats of Lambda DNA and 1 loading buffer as a reference. (Produced in the same way as yesterday). Me and Jo got the opportunity to cut the yeast plugs ourselves today, which was really good! We then started the PFGE at 2.0 V/cm for 4 hours with our new field switching time of 45 seconds. We waited for half an hour, then we turned on the pump, this time was to prevent our liquid Lambda from being washed away.

After the 4 hours, we removed the gel, stained it, drained the buffer and viewed it under UV. This time we saw very dense DNA still within the wells, but fairly good band separation close to the well. Mike suggested that we should seal our yeast plugs with agarose to ensure that the DNA left the wells and began separating. He also suggested that we change our program profile, therefore having an intitial stage for 3 hours at 2.0 V/cm with a field swtiching time of 15 seconds, in order to give the DNA time to leave the wells properly before switching to 45 seconds field switching for the reminder of 24 hours; 21 hours. We then also decided to have a final stage for an hour at 0.6 V/cm, and 45 seconds field switching, until we could get back in Friday morning to check the gel, remove, stain and view it.

Ready to get started tomorrow morning, with our new changes! 🙂

Hey Everyone My name is Amy as I’m…

Amy Thompson / / Uncategorized

Hey Everyone! 🙂

My name is Amy, as I’m sure some of you will already know, and I have just finished my second year as a Biomedical Science student. I haven’t actually started my lab work yet, but you will see me around the lab from Monday 18th June, as I start my UROS project with Mike Shaw. Our project involves the molecular characterisation of clinical Propionibacterium acnes strains by pulsed-field gel electrophoresis (PFGE), and whether this technique is too much of a technical challenge for an undergraduate student. I am therefore very lucky to have Jo Manev, as well as Mike to help me out!

I am really looking forward to getting started with the project, even though I know it is going to be a challenge at times! I hope this opportunity will help me to become more confident in the microbiology/molecular biology lab and prepare me for my final year project, which I hope will be in the field of microbiology/molecular biology. After graduating I am very interested in progressing to PhD study, hopefully within microbiology/molecular biology. Therefore, the opportunity to work along side current post graduate students, lecturers and other students (all of you!) will be incredibly valuable, in terms of gaining first hand experience and advise to help me achieve my aspirations.

As well as blogging on this site with you all, myself, Mike and Jo will hopefully also be producing a video blog of interesting and important milestones during our project. Which should really fun and useful for other students to watch!

See you all soon! 🙂