Category: Uncategorized

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! 🙂

Hey finally round to my second of these…

Dan Firth / / Uncategorized

Hey finally round to my second of these.

The catchup is that we have cleared our plan with Clare for the micro, and have poured a couple of Agar plates and are now waiting on results of our negative control (Blank sponge) hopefully tht will come back as expected and we can begin processing our samples next week.

Learnt how to use the stomacher with Sophie and had a good time in micro, fascinating what some of the guys are doing research on even if some of it is murdering caterpillars!!!!!!!

The fiskerton skull that Jacob is working on is now free of the massive lump of clay, and the skull is nearly clean and ready for analysis. It took 3 days to extract the clay!!!!

Sophie and i also trialed swabbing bones with ethanol to disinfect them, as reccomended by a guy at the university of Indianapolis, glad to say the bones were not damaged in any way (Dread to think what Gillian would have done if they were damaged)

Overall very good time on this research and the things in between like school visits. We had people from two of the three school groups pass out in gen lab 3, starting to think the room is cursed?!?!? I myself believe am getting very good at talking about the bones and how to analyse them, Gillian emabarrased me slightly by testing how to side bones and i failed; so will now work on that for next time.

Hopefully be living in SB209 from next week, so see you then

Hey all My plasmids I transformed and re…

Soph Marie Scanlon / / Uncategorized

Hey all!
My plasmids I transformed and re-transformed from the competent cells I made up on monday didn’t grow overnight unfortunately so I’ve had to start from scratch and made up some DH5a e.coli cells which took over 5 hours to grow today! I’m transforming my cells tomorrow and I can then make some broths up so I can finally do alkaline lysis and gel electrophoresis. Nikki’s helped me to identify where i’ve been going wrong so I’m hoping to finally get some results!
Seeya tomorrow.
Soph 🙂

Evening all Today consisted of A LOT of…

Amy Thompson / / Uncategorized

Evening all!

Today consisted of A LOT of waiting! We started by washing the plugs in 1x wash buffer 4 times, for an hour each! This is to make sure that all the lysozyme and mutanolysin was removed. The plugs could then be stored in 1x wash buffer at 4°C.

We then put each plug into a separate eppendorf tube and washed once with 0.1x wash buffer for an hour, and then once more to ensure all EDTA had been removed, as it will remove cofactors that are very important to the restriction enzymes we want to use next! We then removed 3 plug eppendorfs, and put them into the fridge, in order to compare samples that were treated with restriction enzymes and those who weren’t. We removed the remaining buffer in the other 7, and added 1x restriction enzyme buffer to each, and left for an hour, at room temp. with gentle agitation. After this, we removed the restriction enzyme buffer, and added fresh restriction enzyme buffer, restriction enzyme (SpeI, which cuts at ACTAGT) and BSA (which acts as a crowder to increase conc. and allow increased collisions). This is to allow the fragmentation of our Propionibacterium acnes DNA, which is on average 100kb size fragments. We then incubated the plugs overnight.

Tomorrow we will be finishing off the plugs, preparing a new gel run and running a new gel with our home made plugs and new modifications.

See you all tomorrow! 🙂

The Tale of Steve the Plasmid by Becky…

Steve Peake / / Uncategorized

The Tale of Steve the Plasmid by Becky Lane.

Once upon a time, there was a little plasmid called Steve. He grew up in a cell called the MicroLab, before being brutally extracted by the alkaline lysis we have come to call ‘Becky’. So cold he was when put in the fridge, then so confused when running through the gel, seperated from all the other little plasmids. He was then almost destroyed by Mr Ethidium Bromide, then blinded by UV light, but he was still alive. Cut, he was. Seperated. Everything around him became like jelly as the agarose solubilised. More solutions came to attack from all sides until he was totally alone. Then PCR. Oh dear. The thing he had been dreading the most. It was finally here. Burned alive as he was torn apart, his entrails ripped from him and multiplied, amplified a million times. Surrounded by entrails, he wept. As his world grew cooler once more, he knew it was over. Once more he was placed in a tank, electricity flowwing through him. But he was weak, tooo weak.

Only his entrails would be visible on that machine they call a computer. All else would be thrown away, brushed out of sight, out of mind.

So there he sat in the hazardous waste bin, all alone, forever more…..

The End.

Becky Lane will return in the Tale of the Caterpiller Lady.

Better late then never eh Week 2 Monday…

Jacob Matthew Abbott / / Uncategorized

Better late then never eh? Week 2:

Monday, Tuesday and Wednesday – We learnt the proper techniques on how to clean bones and began work cleaning up boxes of bones we identified as dirty last week. All in all we cleaned 3/4 boxes worth. I also had a chat with Ron Dixon about the Fiskerton skull. He gave me loads of different ways to get the ball of clay inside the skull out.
Thursday and Friday – These two days were research days, I went to the uni library and checked out loads of books on the Iron Age.

Hey all Unfortunately my competent cells I transformed…

Soph Marie Scanlon / / Uncategorized

Hey all!
Unfortunately my competent cells I transformed didn’t grow so I spread plated the cells I grew yesterday again to see if it’s my spread plating technique isn’t correct. I then used the competent cells I made yesterday and transformed them again today using the plasmids pALA1029 C, pOG4 C, large pOG04 and pOG.003 and then spread plated them. Both sets of plates are currently growing in a incubator at 37 degree’s overnight. Really hoping they grow so I can do alkaline lysis tomorrow!
Soph 🙂

Quiet day in the lab today A few…

Deborah / / Uncategorized

Quiet day in the lab today!
A few of the yeast cultures i had did not grow after their first innoculation onto MEA plates. I have re-streaked Mucor, Aspergillus niger and Saccromyces spp onto fresh MEA plates and incubated them at 30C. Penicillin notatum and Aspergillus parasitucus successfully grew and i have re-streaked them onto fresh MEA plates and incubated them for two days at 30C, from this i hope to gain a pure strain of the target funghi. Confirmed Chromobacteruim CV026 and have made a broth culture so that i can make up long term storage stabs and slopes. Also made up a batch of TAE with Rosie and Fran. Now to research exactly what it is and does.

Good Evening Everyone Busy day today Myself Jo…

Amy Thompson / / Uncategorized

Good Evening Everyone!

Busy day today! Myself, Jo and Mike discussed possible modifications to the plug making method when using Propionibacterium acnes. We decided that we would need to use mutanolysin, which aids in cell lysis, as Propionibacterium acnes can be difficult to break into! We also decided to prolong the incubation time after the addition of mutanolysin and lysozyme from 2 hours to 3 hours at 37°C, in order to give them plenty to time to work on the Propionibacterium acnes.

We then began running through our first attempt at making the plugs! We started by adding chloramphenicol to our Propionibacterium acnes TYGE broth (that Mike inoculated for us). Once that had incubated for an hour we centrifuged it at full speed for 5mins, until we obtained a pellet. We then eluted this pellet in cell suspension buffer. We melted agarose and added these together. We then pipetted this into plug molds, which required a bit of practise! (we made 10 plugs) and left them at 4°C, to solidify. Once set we removed them into a universal tube and added lysozyme, it’s buffer and mutanolysin, it was then incubated at 37°C for 3 hours. We then removed the plugs and rinsed the plugs with pure water. We then added proteinase K and it’s buffer and left it to incubate at 50°C, until tomorrow morning.

We will be washing the plugs A LOT tomorrow! 4 times per plug 1 hour each, with wash buffer! We will then be working through the restriction enzyme digestion of the plugs info, and hopefully get ready to set up our first run with our hand made plugs! 🙂

See you all tomorrow!

Hi all Restreaked a few nutrient agar plates…

Deborah / / Uncategorized

Hi all,
Restreaked a few nutrient agar plates, hoping to find pure colonies of the target bacterial cultures that were incubating. One of the cultures didnt need to be streaked, as it was a confirmed bacteria, chromobacterium violeceum. Confirmed as it produces low, convex colonies with a dark purple/metallic sheen (violacein production). This culture, using aseptic techniques, was innoculated into nutrient broth and incubated at 30C for a coulpe of days. Then it will be made into stabs and slopes for long term storage. Researching more on selective agars to confirm more of the cultures on my lab bench. Made up more nutrient agar plates, counted colonies from Sharons Brayford pool project, cleaned lab – quite theraputic. Can’t wait to see what tomorrow brings.

Hey all I made some competent DH5 alpha…

Soph Marie Scanlon / / Uncategorized

Hey all!
I made some competent DH5 alpha E. coli cells today (which took 5 and a half hours to grow to the optical density of 0.350!) and used the colorimeter to see if they had reached the optical density . I have also transformed them so I can do alkaline lysis tomorrow! Still in the lab currently waiting as the transformed cells are currently in the incubator for 45 minutes then I need to spread plate them on to LB agar plates that have ampicillin in them and put them in a incubator over night. I’m using the same plasmids again (pALA1029, pOG4, pOG04, pOG4.003).
See you all tomorrow!
Soph 🙂

Good evening I haven’t really wrote about what…

Sophie :) / / Uncategorized

Good evening! 🙂
I haven’t really wrote about what we have done so far on our project but after reading Dan and Jacob’s entries I can see they have pretty much covered everything.

But in my own words, in week 1 we learnt how to fill out inventory forms correctly when looking over skeletal remains which included noting whether bones were present or absent. This was quite easy to get the hang of after the first skeleton. There were 4 skeletons laid out for us to look at and get to bases with the names of all the bones properly and where they lay in the human structure; so our next task was to lay out a skeleton for ourselves and understand how to differ between left and right bones etc. After getting to grips with the skeletons we started looking into how you work out the stature of the skeleton, by looking at the leg bones (femur & tibia), and how to determine age (using the pubic symphises on the pelvis and teeth if any were present) and to determine sex (using 5 main features on the skull and also the pelvis). Jacob, Dan and I all went to visit St. Katherine’s which is a church converted to a small museum. We went here as a lot of the bones we have in the lab are from St Katherine’s, however, it didn’t prove to be necessary as there wasn’t much information on the actual bones.

Last week was spent mostly doing inventory of all the bones in the lab and deciding which ones needed cleaning and so learnt the cleaning methods, which are quite simple for mud covered bones, being warm water and a toothbrush! But I imagine cleaning densely covered bones is more difficult. The rest of last week was spent doing our own research to prepare for our start doing the microbiology work, such as researching the best sampling method and the best agar to transfer it too along with looking at what types of bacteria are already present on bones and our hands to see if these can cause degradation.

Now comes the exciting stuff and will keep you all up to date at the end of next week!
Have a good week! 🙂

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

Evening All Today myself and Jo came into…

Amy Thompson / / Uncategorized

Evening All! 🙂

Today myself and Jo came into the lab at 9am, and Mike gave us some new stuff to play with! – the newly arrived BioRad plug making kit and restriction enzyme digestion 🙂
We worked through the kit and information provided, made some notes and researched anything we didn’t understand online and through a discussion with Mike. We then produced a new COSHH assessment for our new procedure of making plugs and researched any modifications that journals mentioned when using Propionibacterium acnes specically.

We then made up a stock solution of Chloramphenicol (90 mg/ml), that we will need during plug production. It is a antibiotic, that is bacterial static and stops replication. We will also be using lysozyme, to break down cell membranes, Proteinase K, to break down protein and restriction enzymes, to cut up DNA specifically. Each also has a specific buffer, this is due to enzymes specific nature (pH, Cofactors). We also discussed why it was important to remove remaining EDTA, as it removes cofactors, which are very important to enzyme activity! We also discussed how obtaining the proper cell concentration within the plugs is most likely to be the most difficult part of production. In order to get the most accurate concentration we will be using an OD and CFU/ml against time to produce two sigmoid curves in order to work concentration out.

Excited to start practising these new techniques, so bring on tomorrow morning! 🙂

Right have been a little late setting this…

Dan Firth / / Uncategorized

Right have been a little late setting this up so here goes,

My names Dan and i’m working on a UROS project alongside Sophie Webber, under the superfision of Gillian and Clare. The project is looking at whether or not wearing gloves when handling skeletal material matters. Obviously in forensic cases gloves should always be worn, but with archaeological remains there is great debate about the need to wear gloves. Hopefully by the end of the project we will be able to see whether or not there a scientifically significant transfer of microbiota from handlers to the bones if not wearing gloves. If there is a significant difference in the quantities of microbiota on skeletal material when not where wearing gloves (In comparison to wearing gloves), the next step is to see whether or not the microbiota transeferred from hands actually degrade the bones themselves.

Thats the basics behind our project. now to bring you up to speed with whats happened in the first 2 weeks.

In the first week, i met with Gillian, Clare and Sophie to discuss the project and what we would be doing. Gillian set us off familiarising ourselves with the bones of the body- learning their names, their anatomical positions, and how to sex, age and obtain stature estimations from the bones concerning the individual- this was a great start point and also really boosts my chances of passing the anthropology exam in October/November.

Following this we were then asked to identify which bones in the universities skeletal collection required cleaning, and then to clean those bones. Cleaning bones is a slow process, but oddly hypnotizing so time flies by.

So thats the important stuff thats happened over the past 2 weeks, which doesn’t sound much but the time spent just examining the bones and learning from Gillian has greatly increased my knowledge. Next week Sophie and i will be joining you in the microlab and the project can really get underway.

The two of us have been working closely with another of Gillian’s UROS students, Jacob Abbott, who has been tasked with cleaning and analysing a skull found in Fiskerton LIncolnshire, that is believed to be Iron age!!!!!! How cool is that!!!!!!!! Unfortunately he first has to work out how to remove a huge lump of clay from inside the skull without damaging it, i wish him luck with that.

So i think i have said everything that needs to be said, i’m really enjoying my project thus far, and it staves off the dull boringness of being alone in Lincoln for the summer hols. See you all next week hopefully

Busy day today Started the day going through…

Deborah / / Uncategorized

Busy day today,
Started the day going through wednesday and thursday’s results with Rachael, so she knows where we are with our cultures, and so we can plan what we’re doing next. Taught Hammad how to do water filtration, made up 950ml of M17agar base with 10g Lactose suppliment (suspended in 100ml distilled water), both were autolaved before mixing. After lunch Rachael poured the plates. Made up 2800ml (7 x 400ml) of LB agar and poured plates with five of these. The other two were left for Nicola to add ampicillin and then pour the plates.I also made up 250ml of 0.85% w/v NaCl, this also needed to be autoclaved. Made up two stabs and slopes with the PS aeruginosa broth that i incubated overnight. Counted colony cultures from Sharon’s Brayfod water project.

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! 🙂

A really successful day Yesterday i streaked 4…

Deborah / / Uncategorized

A really successful day!
Yesterday i streaked 4 Pseudomonas CFC agar plates with cultures of PS aeruginosa.Today the plates showed excellent growth and the cultures glowed florescent green. Gram staining showed that the cultures were Gram negative and rod shaped. All this was evidence that i had a pure strain of PS aeruginosa, I innoculated nutrient broth and incubated it overnight at 37C. Tomorrow i will make up two stabs and two slopes. One confirmed bacterial culture down twenty seven more to go!!! Using the Qcapture pro (microscope attached to a computor) i saved images of my Gram stained cultures to usb, hopefully i will post pictures soon.

Hello Everyone Sorry I Haven’t done a blog…

Rosie Myers / / Uncategorized

Hello Everyone! Sorry I Haven’t done a blog in a while… But i will start doing them now!

So here is a basic low down on what is going on in my project!
First I had been extracting E.coli from the Brayford and growing them on MSLA agar plates. These are the red plates and they will only grow the Ecoli from my sample as yellow, well rounded colonies. With the whole streak plate/broth/agar situation.. I have never really had much practice with it.. But now i know how to do it all! 🙂 (Thanks BioMeds)

In the last couple of days I have filtered 100ml of Brayford Water onto MSLA plates containing 100ug Ampicillin. This is a very high concentration of antibiotic, therefore the colonies I obtained on these plates were very resistant and pure. I then proceeded to make several LB broths with Ampicillin with the colonies from the plates. Today I used the QIAcube with my plasmid broths, and I also did alkaline lysis by hand and ran the samples side by side on a gel to observe the differences/similarity between the different methods and to see whether I had any plasmids and to interpret the banding patterns seen on the gel.
I found that in 2 out of my 6 samples I had a very distinct interesting band pattern occuring, so I will keep these and do futher work with them later and all the rest showed nothing so they have been disposed of!
I will now start making agar plates with different antibiotics on – starting with Ceftriaxone.
Once I have gathered all of this information from the different antibiotics I will see the range and frequency of plasmid stability systems present in plasmids conferring antibiotic resistance in the Brayford allowing understanding of their persistence in the environment!

This will also enable me to have an understanding of plasmids from E.coli that confer antibiotic resistance, focussing on the CTX-M ESBL gene which is found in E.coli which are resistant to third generation cephalosporins such as Ceftriaxone and cefotaxime. This is why I will be doing the above procedure just with these antibiotics on the plates and in the broths!

See you all tomorrow 🙂 Xxx