Hi today luke did water filtration with water…

Deborah / / Uncategorized

Hi,
today luke did water filtration with water from the Brayford and innoculated nutrient agar plates with the filtered discs and incubated them at 30C for two days. The mannitol salt agar plates that had been spread plated with water from the Brayford showed large yellowy mucoid colonies, this was a positive for a Staphylococcus species. Using a sample from the mannitol salt plates and from the E.coli luke streaked the day before, Sharon and Luke performed Gram stains to compare the colonies. We then made up 1000ml of 50X TAE, and a 1% agarose gel for Steve. I have confirmd two more of my bacterial cultures, hopefully tomorrow i can inoculate them into broths and then into stabs and slopes.

Hi all firstly sharon luke and I collected…

Deborah / / Uncategorized

Hi all,
firstly sharon, luke and I collected a water sample from the Brayford, then we each spread plated a couple of nutrient agar plates with 0.1ml of the Brayford water and incubated them at 30C for two days.Then Luke streaked a couple of nutrient agar plates with E.coli and incubated them overnight at 37C. Luke spread plated a couple of mannitol salt agar plates with the Brayford water. We then watched Rosie innoculate her plasmids with antibiotics as she explained to us what she hoped to find. Explained water filtration to Luke as this is our task for tomorrow.

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 🙂

Hi all Interesting day started the day by…

Deborah / / Uncategorized

Hi all,
Interesting day, started the day by making 3000ml of TAE buffer. Next i ran a PCR test with a sample of my hair. When we looked at the gel under the UV some showed two bands and some showed just one band, either i’m a boy or luke is a girl. Think we may need to redo the test and look up which banding type (1 or 2) shows a male or female. Researched a few of the bacterial cultures on my bench, some are same species where an indole test is the only way to differentiate them.

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 🙂

Hello Today i started producing competant cells and…

Rosie Myers / / Uncategorized

Hello!

Today i started producing competant cells and doing transformation.
Competent cells were made by first creating an LB broth by placing a colonie from a DH5 alpha plate into it, and having it incubated and shaking for around 5 hours! Once my cells were at the right optical density, I could proceed. As it happens they grew really fast and well so thats always a good start! Once I had centrifuged my cells down multiple times, placed them on ice, and added amounts of CaCl2 i ended up with cells which were then competent and capable of taking up plasmid DNA. The competent cells could be used immediately for transformation and (hopefully) uptake my plasmid DNA which i have obtained from my previous weeks in the lab!
I then began transformation where i added my different plasmid DNAs with some competant cells and proceeded to heat, cool, heat, cool etc! It is a very long process! But it enables the cells to recover and the antibiotic resistance of the plasmid to express itself 🙂
Basically with the solutions i then had, I spread 100ul from each one onto LB agar containing ampicillin, and if there are colonies present tomorrow morning then this means that the cells have uptaken my plasmids, and that i do have plasmids stocks that are resistant to amicillin! If i do i will have lots of things to play with 🙂 If not… i shall start all over again. 🙁
I have also spread a positive control, so that if it doesnt work tomorrow this will tell me if it was something in my methodology that was wrong or just that the plasmids in my DNA samples are not resistant to ampicillin, and it was probably just the chromosomes 🙁

Any questions feel free to come and ask! :):)

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 🙂

Hi all Checked Lactobacillus plates Two do not…

Deborah / / Uncategorized

Hi all,
Checked Lactobacillus plates, Two do not look like the other three, after Gram staining a sample colony from all the plates, two had Gram positve small cocci. The other three showed long rod shaped gram negative chains, ths was more like what i expected to find. To be sure, i restreaked these onto M17agar with added Lactose and incubated them overnight at 37C. In preparation for tomorrows PCR tests i made up the solutions needed, working out quantities of HCl and Tris-HCl in 200mM. Microbiology is fun but maths makes my head hurt!!! big thanks to nicola for her little equation, i now can work molar solutions out but i think more practice is definately needed

Hello So as Dan has already mentioned last…

Sophie :) / / Uncategorized

Hello!

So, as Dan has already mentioned, last week we spent a few days in the micro lab preparing nutrient agar plates to test our negative control of the polywipe sponges.
The polywipe sponges are out of date (Oct ’11) and therefore we needed to do a negative control to see if we could still use them to swab our bones.

We tested out 3 ways to put our sample onto the agar plate, a simple 0.1ml spread plate, another plate wiping the sponge itself onto the plate and the third way was by filtering the rest of the ringers solution onto a filter paper to place onto the agar. We learnt how to use the stomacher which is pretty simple so will be able to use that in the future for our project samples.
After incubating the plates at 37degrees for 24hours we could see some small colonies on the plates!! NOT WHAT WE WANTED! So then over the weekend we incubated them again this time at 25degrees to see if anything else grows.

Unfortunately it did 🙁 and now we can’t use the polywipe sponges and have to wait for new ones to be ordered in!!!

Today we checked up on our plates and recorded our observations, they seemed to be fungal growths on the plates so we sub-cultured them onto an MEA plate for curiosity as to where the fungi came from and possibly identify it. This has been incubated at 37degrees again and we will look at this tomorrow to see if anything has happened!

Evening all x Yesterday did a PCR test…

Deborah / / Uncategorized

Evening all x
Yesterday did a PCR test with my cheek cells, mouthwash was yuck! but good news is i’m human. Today was a day of filling in COSHH and risk assessment forms, definately NOT a fun part of my day, but necessary. Confirmed M.luteus so made up stabs and slopes, Chromobacterium CV026 also confirmed, made into stabs and slopes. Made up a batch of TAE and another litre of 50x (Tris base, glacial aceitic acid, EDTA and made up to 1L with distilled water). The main thing i learned, is to not go to lunch and leave the tris to dissolve. increased pressure made it near impossible to remove the stopper, fortunately we are scientists – heating the glass whilst running cold water on the lid, hey presto, the stopper comes off.

Hey All Not much to report today myself…

Amy Thompson / / Uncategorized

Hey All!

Not much to report today, myself and Jo got into the lab for 9am and tried to film our videoblog! Although someone couldn’t remember how to speak properly, so it took us atleast an hour to finally get some film we were really happy with 🙂

While we were filming we removed our gel, stained it and when we were finished viewed it under UV. We saved the image, as Mike wasn’t in today.

Third week done already! See you all on Monday 🙂

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 🙂