Category: Uncategorized

Groundhog day Today is still Monday no overnight…

Deborah / / Uncategorized

Groundhog day!!!!
Today is still Monday, no overnight growth again. Think C2110 is mocking me. I have repeated everything once again but i have tried to trouble shoot as to why nothing is working. Once i made thre cells competant i plated a sample onto LB agar plate and a sample onto LB agar with amp50. Then i continued on to the transformation stage, i then plated a sample onto LB agar plate and onto LB amp50 agar plate. I have also restreaked a fresh nutrient plate with C2110. Hopefully things will grow overnight, if not maybe i can work out at what stage things are going wrong.

Hello Today i began by removing the gel…

Amy Thompson / / Uncategorized

Hello,

Today i began by removing the gel, staining it and viewing it. The gel was much cleaner than before, and the low range PFG marker showed very clear bands, while the restriction enzyme treated plug showed some separation but blurring as before. This confirmed that the problem lies with the plugs, not the settings. Which will hopefully be cleaned up, after DNA conc. is increased in the new plugs.

I began plug making as the Propionbacterium acnes 6919 strain sample had nearly grown enough. Mike suggested i use 2 tubes of 10ml Propionbacterium acnes 6919 strain sample, add chloramphenicol and centrifuge down all 10ml of both samples. This produced a large pellet that i then eluted in cell suspension buffer and combined with agarose. I then pipetted this into 5 plugs molds, and left these to solidify. Once this had happened, i pushed them out into a universal, added lysozyme, buffer and mutanolysin and left to incubate at 37C, for 5 hrs. I then removed this, and added proteinase K and buffer, and incubated overnight at 50C.

Tomorrow i will begin washing the plugs, and treating them with SpeI.

Hey All Didn’t come in till late today…

Amy Thompson / / Uncategorized

Hey All!

Didn’t come in till late today, to allow Propionbacterium acnes 6919 strain sample to grow, however the sample hadn’t grown enough, so we have left it to grow till tomorrow.

Meanwhile, i removed the plugs from speI, washed them in 1x wash buffer for 30 mins, then incubated them in TAE 0.5x until i had made and set a new gel. I used the modified comb as before to create an extra large well, so i could use 2 plugs in 1 well, as the plugs are from an old batch and therefore still contain minimal DNA.

I then ran the PFGE for 15hrs, at 6V/cm, with 1-12sec switch intervals. Mike also suggested using an increased amount of buffer in a large container below, and pumping buffer through the PFGE to replace buffer as it left the PFGE back into the container. This will hopefully keep all the buffer in the container as cold as possible, reducing smearing and making bands clearer.

Tomorrow, if the cells have grown enough, i can start to make new plugs, with our modifications.

Hey Yesterday i grew C2110 E coli to…

Deborah / / Uncategorized

Hey,
Yesterday i grew C2110 E. coli to an OD of 0.35. Three hours later!! I made the cells competant, then i transformed them, leaving them in the 37C shaker for 45 minutes. Once this was complete i spread plated 0.1ml onto an LB agar plate containing amp50 and left the plates to incubate overnight at 37C. Unfortunately this morning NOTHING had grown. very frustrating, so today is now monday and i’m repeating everything again!!

Hello Everyone First day back in the lab…

Amy Thompson / / Uncategorized

Hello Everyone!

First day back in the lab! Me and Mike had a quick chat about what i wanted to do next and made a plan for this week, and possible plans for my final few weeks in the lab – it’s gone soo fast!
Mike re cultured the Propionbacterium acnes 6919 strain samples to allow them all of today and tomorrow morning to grow. Meanwhile, i treated my 3 remaining plugs with restriction enzyme – SpeI, which needs to incubate overnight. I also prepared and autoclaved 3L of 0.5X TAE buffer ready for running a gel with the restriction enzyme treated plugs and the low range PFG marker, using the settings we decided on last time i was in the lab – 6V/cm, 15hrs, 1-12sec Switch Intervals and a 1.4% gel.

Tomorrow will be a late start, to give Propionbacterium acnes 6919 strain samples as much time as possible to grow, before i begin making new plugs with our decided on modifications and setting up and running a new gel with the restriction enzyme treated plugs and new settings. Hopefully we will get some clearer and just as positive results! 🙂

Hey all Yesterday i took the overnight broths…

Deborah / / Uncategorized

Hey all,
Yesterday i took the overnight broths for the two plasmids and completed another dilution series and spread plated the ones from -5.5 and -6. I incubated these at 37C overnight. Then i took out the T0 plates and counted the colonies, the ones that were around 100 i replica plated them onto an LB amp100 agar plate and then olnto an LB agar plate. These were then incubated overnight at 37C.
Today started out with me making more LB agar and LBamp100 agar plates, and making up 3x50ml LB broth, ready for monday morning. Then i autoclaved my velvets ready for next week. Once this was completed the science could start. I counted Tend plates and replica plated the ones with around 100 colonies, same as yesterday i replica plated 1x LBamp100 and 1xLB agar. The last job of the day was to count the colonies from yesterdays replica plating and record them. I also re streaked my C2110 so that i will be working with a fresh culture on monday.

YAY got growth finally can start I took…

Deborah / / Uncategorized

YAY!! got growth, finally can start. I took four colonies from each of my culture plates and made them up into broths using LB broth and adding ampiillin 100. Then i placed them in the shaker at 37C, until they grew to an OD of 1(0.8-1). then i set up a dilution series, labelling eppendorfs A,BC,D -1 to -5 adding 0.9ml NaCl and 0.684ml into the eppendorf marked -5.5. Once the broths reached an optical density of 1, 0.1ml was added to each eppendorfs with the exception of the -5.5 as 0.316ml broth was added. From the 10 -4 eppendorf, 100microlitres was innoculated into 10ml of LB broth and incubated overnight at 37C. The -5 and -5.5 dilution series were spread plated onto LB agar plates and incubated overnight at 37C. This was repeated for pOG04 NJC and pOG04 DRW plasmids. Cant wait for tomorrow.

Really quiet day in the lab today was…

Deborah / / Uncategorized

Really quiet day in the lab, today was the first day of my Uros, plasmid stability experiment. Started the day by growing E.coli C2110 to an OD of 0.35, tough to guess when this happens, 0.38 was the final reading. Whilst waiting for the cells to grow i then innoculated another flask of LB broth with C2110 and placed it in the 37C shaker.The next task was to make the cells competant and then transform them, using the plasmids pOG04 NJC and pOG04 DRW. The final step was to innoculate an LB agar plate (containing Ampicillin 50) and incubate overnight at 37C.

Hey Today was a strange day Did my…

Deborah / / Uncategorized

Hey,
Today was a strange day, Did my final Tend plate count for the week. I inputted all of my weeks figures into a table and Nicola showed me some impressive maths to get to the percent stability of the plasmid used, in this case pOG 04. The percentage was 63, which was waaay out from the expected 3-5%. I am going to redo this plasmid next week and hopefully (wearing my lucky pants) everything will work out as expected. Did a little research into my eight final bacerial cultures still on my bench, i will not be defeated!!!
Also sad to see Sophie and Dan finish their project, both will be missed. Also going to be a little quiet without Amy and Jo whilst they’re n hols. Happy summer guys x

So the last post on the blog by…

Sophie :) / / Uncategorized

So…the last post on the blog by me! Finally finished our UROS project today! Excited to have a bit of summer left! Had an amazing time and it’s been a great experience and i’ve learnt alot. I’ve learnt all the bones in the human body, and many anthropology skills and techniques to age sex and determine stature and these skills i will keep with me to help in my final year! I’ve also met lovely people in the micro lab that i probably wouldn’t have got chance to if i hadn’t done this project so for that i am thankful! Being able to work with my peers and lecturers has been a good experience and being able to work in a lab for a long length of time!

On to the project results…this week we swabbed the bones after being cleaned with ethanol and then we handled one set of bones with gloves on and one set without gloves and swabbed these too to come to some kind of conclusion as to wearing gloves affects contamination on the bone. The results mainly pointed in this direction with the swabs taken after handling without gloves gave more bacterial growth than that with gloves on.

We weren’t able to identify any of the bacteria in this project but it will make for an interesting project next summer maybe!

Thanks again everyone involved, its been great! 🙂 Enjoy your summers!!!

Hey all Today we removed stained and viewed…

Amy Thompson / / Uncategorized

Hey all!

Today we removed, stained and viewed our gel. We saw some very promising results! Although the separation wasn’t great, we saw a large blurred band near the bottom of the gel, near the middle of the low range marker bands present (which unfortunately were quite blurred and difficult to see). This confirmed the presence of DNA in the SpeI treated plug, if not very much, as estimated previously. However, enough DNA was present within the whole plug to show separation, confirming that our plug making procedure and settings were near enough on the right track!

Given the set backs we have been experiencing with the plugs this week and last, this was a real boost to get us excited to start putting our modifications into practise for plug making – increasing cell density/DNA and slightly modifying the PFGE settings – increasing the gel conc. from 1% to 1.4% in order to condense the ladder bands nearer the top, allowing further reference to the Propionbacterium acnes restriction enzyme treated plugs near the middle of the ladder.

We filmed our last videoblog for a week today, as i am away from the lab next week, this will also be my last blog for a week 🙁
Jo will also be away for the next 3 weeks, so we will not be able to put these new modifications into practise just yet! But i am very excited to get started, on what will hopefully be modifications that get us our best results yet for restriction enzyme treated and non restriction enzyme treated plugs on PFGE 🙂

See you all soon!

Hey Started the day by making 400ml of…

Deborah / / Uncategorized

Hey,
Started the day by making 400ml of LB agar and 200ml with AMP100 and 500ml of LB broth. Then i counted the T0 replica plates i did yesterday. Next i counted yesterdays T end plates and chose the ones with approx. 100 colonies and replica plated them onto LB agar and LB agar with AMP100. Then went to the presentation for poster presentation and project write up. Then i streaked a fresh LB agar plate with C2110 colony and then another LB agar plate with DH5alpha (for Rosie) and incubated them at 37C overnight, so that we have fresh colonies to work with next week. Nicola gave me my plasmids, popped them in the fridge. Nearly all prepped for next week. Also managed to make up stabs and slopes of E.coli e0791 that i brothed up yesterday. Also BIG thanks to Dan for his anthropology presentation and showing me the difference between boys and girls (on the skeletons). Very interesting seeing the ossicles and learning how to age and sex the bones.

Hello Today we removed our latest gel stained…

Amy Thompson / / Uncategorized

Hello,

Today we removed our latest gel, stained it and viewed it. Unfortunately we didn’t get very good results – there was alot of blurring on one the low range PFG marker samples, lack of clear band separation on the low range PFG marker samples and no expected band just below the wells for the non restriction enzyme plugs.

We decided that this was most likely due to lack of cell density, and therefore lack of DNA which that could be seen on the gel. We discussed that this could most likely to be fixed by allowing the Propionbacterium acnes samples to grow longer and therefore increase the cell density and DNA, and possibly by centrifuging more of the sample, to reach a higher conc. We also re researched info on cell density within plugs in the manual and journals – we found that the optimum OD was around 0.8-1.0 and we had been working with 0.24. Because of this increase in cell density/conc. we will also need to increase our lysozyme, mutanolysin and proteinase K to compensate.

Mike asked us to run a standard gel, in order to see if there was actually any DNA present at all. We ran a 0.8% gel, for 2 and half hours at 120V/cm with a 1kb ladder. We saw very faint separation, that did confirm that DNA was present, but at very low density/conc. as expected. This also confirmed our modifications which we will put into practise when i come from holiday on Monday 6th August.

After speaking to Mike we decided to attempt to run a new PFGE gel using the whole Propionbacterium acnes restriction treated plug, in one well, in order to use as much DNA as possible, and hopefully see some separation showing multiple bands due to lysis. We ran this 1% gel at 6V/cm, for 15hours with 1-12 sec switch interval ramping, using a whole Propionbacterium acnes restriction treated plug and the low range PFG marker.

Tomorrow we will be viewing this gel and filming our latest videoblog! 🙂

Hi all Started day by completing my serial…

Deborah / / Uncategorized

Hi all,
Started day by completing my serial dilutions from yesterdays broth culture, I spread plated the dilutions of 10-6 and 10-6.5 on LB agar and incubated them overnight at 37C. Then taking the eight plates from yesterday I counted the colonies and chose the four with around 100 colonies. I then replica plated each of these onto a further two LB agar plates (one containing Ampicillin 100). These were then incubated at 37C overnight. I then also broth cultured another confirmed strain of E.coli (e0791).

Evening Today we washed our plugs as before…

Amy Thompson / / Uncategorized

Evening,

Today we washed our plugs, as before in 1X wash buffer 4 times for an hour each. We then separated our 5 plugs into separate eppendorfs, and put 3 in storage in fresh 1X wash buffer. To the 2 remaining plugs we added 0.1X wash buffer for an hour.

To one we removed the wash buffer and added 1ml of Spe1 buffer to saturate for an hour. We then added fresh Spe1 buffer, Spe1 enzyme and BSA, and incubated overnight at 37C.

We set up a new gel in order to run the unrestriction treated plug, to ensure that the DNA within the samples are viable for use. If we see one large band just below the wells, that is as clear or clearer as our best previous gel run, then we will run a gel with the same settings, but with restriction enzyme treated plugs.

We made up 3L of TAE 0.5X and a 1% gel. We removed the wash buffer from the other plug and added 1ml of TAE 0.5X buffer to saturate it. Once the gel had set, we cut and placed 2 non restriction enzyme treated plug sections and 2 sections of the new low range PFG marker into the wells and sealed with agarose. We ran this gel at the low range PFG marker settings: 1% agarose gel, 15 hours, 1-12 second switch interval ramping and 6V/cm.

I also filmed with Sophia and Barney, regarding their experiences in the lab so far! 🙂

We will remove this gel tomorrow, stain it and view it. This will help us to decide whether to run the restriction enzyme plug, with the same low range PFG marker settings.

See you all tomorrow!

Hi Started the day Gram staining E coli…

Deborah / / Uncategorized

Hi,
Started the day Gram staining E.coli a strain i dont have, this is another cultue confirmed so i then made a broth which i am incubating overnight at 37C. Spent most of the day waiting for cells to grow to an OD of 1, though 0.8 – 1.5 would be within range. Nicola went through serial dilution with me and showed me how to do the first set, i then completed the following three, broth culturing -4 dilution and spread plating -5 and -5.5 dilutions onto LB agar plates and incubated them overnight at 37C. Hopefully tomorrow we will have enough growth to count colonies (100 would be nice) then we can do replica plating. Had another Mike Shaw masterclass and debate (loving lab life xx).

So i keep forgetting to post but Dan…

Sophie :) / / Uncategorized

So, i keep forgetting to post but Dan has been up to date which is good so you can get the idea of what we have been doing!

Week 4 consisted mostly of waiting for our supplies to come in again and taking student as producer literally by experimenting with cotton swabs on some of the bones after handling to see how long the bacteria maintains on the bones. The results for these came in all shapes colours and sizes and so we were unsure if they were viable and not contaminated. We also helped out an an open day and workshops teaching the students how to age, sex and determine stature of the skeletons.

Week 5 we continued the experimenting into the cotton swabs and the bacteria living on the bones. However Wednesday we actually found all our supplies and so the micro side of the project finally began!!!
We plated up agar and made ringers in prep for our swabs. Starting with swabbing the bones with the polywipe sponges for a background check to see what was already on the bones, doing 5 repeats of 5 bones so a total of 25 nutrient agar plates, these were incubated at 37degrees for 48 hours and looked at again on Friday! Obviously as Dan has already said, the results were weird! research for you. They could have been from contamination, if not then our bones have lots of different types of bacteria/fungus present!! We sub-cultured most of the plates onto several different types of agar and we also took samples and gram stained them to see if we could identify what we had…this left us even more confused!

Now we are in our final week of the project and yesterday we cleaned 5 of the bones with ethanol (50%, 75% and 95%) and let the ethanol soak in to destroy bacteria, then swabbed with the polywipe sponges and placed in ringers solution, each plate had 0.1ml pipetted on and there was again 5 repeats for each bone. The same bones were then handled with gloves for 5 minutes and then repeated the swabbing steps to see what grows on bones handled with gloves. Today we made up 60 more agar plates for use tomorrow when we will clean another 5 bones and handle these with bare hands and see how they differ, like the project is called….gloves or no gloves?

We will have to wait and see!!