8th June 2016 – Day 13

Wednesday:

29˚C in the lab today, we had everything open, windows, doors, everything and it was still too hot. The university was having some form of a sports day and we could hear all the cheering coming in through the windows. We all kept getting a fright as the horn sounded throughout the day, creating a nice atmosphere and keeping us on our toes despite the heat of the day.  (We all looked for an excuse to go down to the phd labs, or the room at the back where we get our ice from, the rooms with the air conditioning .... just so we could cool down a bit.) 

Today we finished off the third protein purification, taking the same samples as we went along to run on a gel. 

We concentrated the third eluted protein the same way we did the Sufu and it was applied to the GF column.

We really knew what we were doing today as we have done it so many times this week, this made things a little more straight forward which was a good thing because of the heat.

7th June 2016 – Day 12

Tuesday:

We analysed the Sufu fractions from the filtration column on a gel. We then pooled the fractions from the GF column (concentrated and quantitated, aliquoted and flash frozen).

The column was washed with water and ethanol and we had to change the size of the column for the second protein which is much smaller in size.

The Gel Filtration Column

The Histag purification was completed with the second protein and samples were taken at each stage and run on a gel.

Looking at the gels, most of the proteins for both were found to be insoluble and we decided to do instead of do can you say run the cultures again but this time incubate them at 18˚C instead of 37˚C overnight. So we began again, making fresh cultures for all and a new broth for the giant culture flasks. We decided to go ahead with the purification of the third protein which we did and incubated overnight again with rotation.

I keep thinking its Thursday, we have done so much in the last 2 days that I feel like it should be the end of the week. I guess the heat doesn’t help, the labs have been incredibly hot (the weather report lied, they said it would rain, it never did, we need just a little rain to cool us down).

6th June 2016 – Day 11

Monday:
One volunteer down today, gone for a whole week… the lucky person has gone on holiday!!

The rest of us prepared 50ml Lysis Buffer (it doesn’t help when you forget the protease Inhibitor tablet the first time round!). The Sufu that was grown in the large flasks last week was harvested today (the same way as before, in the chilled centrifuge), the pellet was frozen at -80˚C for an hour and then thawed and resuspended in 16ml lysis buffer.

The -80 freezer, always makes my hand go bright pink!

While we did this we also made 4 gels, we made doubly sure that we added everything, and decided to use the expensive gel equipment from downstairs because we were told that it would reduce the cross contamination between wells and produce more clear results… so we shall see.

Once the pellet had been resuspended, we sonicated the pellet with the large probe (this uses sound waves to break open the cells releasing any proteins inside, it makes a horrible high pitched noise… but it meant we got to wear some very awesome looking bright yellow noise cancelling headphones…we looked very cool). We took a small sample to run on the gel and put the rest of the Sufu sample in the centrifuge, 1700rpm for 20 minutes. The supernatant was filtered off and the pellet was resuspended in TBS buffer (a sample taken from both for the gel).

The sonicator and those cool headphones

We did a histag-purification of the supernatant using nickel beads, the beads were washed well with buffer A and then the supernantant was added and left to incubate with rotation for an hour at 4˚C.

We used this time to take our lunch break and we decided to take it outside, as it was a nice, hot, sunny day.  This turned out to be a bad idea, I had the worst headache in history… for the entire afternoon and my lunch partner ..... well she was convinced she had heat stroke, she was rather pale to be honest. This made the afternoon slow but we soldiered on and still manged to finish everything we needed to.

We carried on with the purification, we poured the incubated Sufu sample into a column, we took all of the necessary steps to collect the flow through, one wash, the elution and then we resuspended the beads in buffer B (elution buffer). We took a sample from each of these and ran them on a gel.

The elute was concentrated using a 50K cutoff concentrator and applied to a gel filtration column. The same process was done to the second protein and it was left to incubate with rotation overnight (we will carry on with the purification tomorrow).

3rd June 2016 – Day 10

Friday:

Buffer day!! We made all of the buffers and solutions we are going to need for next week (in preparation for what we are planning to be a very busy week.)

1 litre of Buffer A – wash buffer

1 litre of Buffer B – elution buffer

One would think these buffers would be easy to make by following the instructions, but for some reason we all kept getting confused, it took us 3 attempts to get it right (at first we added the concentrations, instead of the volumes and then we put the opposite volumes of Imidazole in to each….really its like we were all having a momentary lapse of reason......they are only buffers). These were both pH’d to pH 7.6 using concentrated HCL. The pH meter had to be calibrated beforehand between pH 10 and 7. These will be needed for the pull down assays next week.

2 litres of gel filtration buffer

1 litre H20

1 litre 20% ethanol (made from 100%)

These last 3 solutions were filtered and degassed, it took a while but it needed to be done for the gel filtration column, it can’t have any bubbles (although who doesn’t love bubbles).

The column is stored in ethanol to stop bacterial growth, so it is washed with water and gel filtration buffer (this is to prepare it for use next week).

It's been such a busy two weeks, but I have really enjoyed every minute. My poor feet are looking forward to some downtime or should I say ''up time'' and a bit of rest this weekend, but I am really excited to see what next week holds. 

Day 9 – Blot Results!!

2nd June 2016 – Day 9

Thursday:

Western blot continued… after the membrane was washed with blocking buffer overnight, it was washed twice with 1x TBS for 10 minutes each. During this time more blocking buffer was made, 10ml for each membrane with 10ul of the corresponding antibody. It was rocked for an hour in the primary antibody. While the membranes incubated in antibody we made some TBS/Tween/Triton buffer after which each membrane was washed twice with this buffer and then twice with TBS/Tween/Triton. Each membrane was then incubated again for an hour in secondary antibody. The same antibody can be used for each membrane as these are not protein specific but can bind to any antibody. Each membrane was washed twice again with TBS/Tween/Triton buffer and then twice again with TBS buffer. Once we had completed this we incubated membranes for 2 minutes each in chemiluminescent reagent and detected the proteins using the pixi (fancy machine down stairs, my favourite).

Pixi (the fancy machine)

We had stuck the gels into de-stain and then photographed each one (this was just to make sure that all the proteins had been transferred to the membrane, a double check if you will).

The cultures from the conical flasks (made yesterday) were harvested by centrifuging for 20 minutes at 8000rpm and 4˚C. The pellets were fished out and frozen in the -80˚C freezer.

1 conical flask was prepared with LB broth and autoclaved in preparation for the streak plate culture, Sufu, then a single colony was taken from the streak plate and was cultured, induced with 0.4mM IPTG and incubated at 18˚C overnight.

I've known the theory of how to carry out a Western blot, so to actually do one start to finish in the lab was very exciting (Go me! Whoop!)

1st June 2016 – Day 8

Wednesday:

Today was the first day of many western blots! (A process of detecting proteins using antibodies). We placed all 4 gels (that we made yesterday) into the tanks, and loaded all of the samples onto each gel. These were run at 100V and 180V, for 1 hour and then 2 hours. 

I made 3% blocking buffer in 1x TBS (this was just milk powder and water, first the potatoes now milk, what next….?.... I am hoping for something cake related). 

Some of the buffers and reagents needed for a WB.

Once the gel had finished running, we needed to transfer the proteins to a nitrocellulose membrane. (The order for transferring: mesh, filter, membrane, gel, filter, mesh, each mesh was surrounded by a plastic cassette). Each was dipped in 1x transfer buffer. The tank was filled with transfer buffer and the assembled cassettes for each were placed in the tank (it holds 4 and therefore each one fits in nice and snug). The transfer tanks were run at 100V, 350mA for 60 minutes. The current didn’t get high enough and there was no visible ladder on the membrane as the frozen ice packs were placed in the wrong position so we had to remove these. We took the cassette apart and soaked the sponge pads and membranes for 15 minutes after which we ran the transfer for a further hour. The membranes were removed, washed with TBS twice for 10 minutes and left to soak in blocking buffer overnight.

After the proteins were transferred from the gels to the nitrocellulose membrane, we put all of the gels in Coomassie blue stain (so we could look at it later).

Gels put into stain.

The overnight cultures we did yesterday were transferred  to a (100ml) conical flask, with 100ul KAN. I showed the volunteers how to make more IPTG (100mM) and how to filter sterilise, by teaching this process it helped solidify it for me. After some time, the OD readings were taken, at 0.573 OD reading they were transferred to the large flasks (you know…. the really big ones). 20ml of culture into the giant flask with the corresponding antibody. It was then incubated for 2 hours. After the 2 hours had passed the OD was measured. It was not high enough and so it was incubated for another hour and 30 minutes, after which the OD was measured at 0.521. The IPTG was added to each culture and incubated overnight.

The western blot was trial and error and we didn’t expect that it would work straight away. We are mostly preparing and practising the protocol for the next few weeks when we really need it. We had to juggle both culturing and western blots throughout the day….(quite challenging)….but what a great day (happy dance).

31st May 2016 – Day 7

Tuesday:

Two new volunteers in the lab today, and Amy charged me with showing them the ropes and overseeing things. I know them both well from class so at least we are all familiar (but one thing I have learnt today is I definitely like being in charge!).

We didn’t do anything new today, it was all similar methods to last week, but it allowed me to build some confidence as I knew what I was doing and what the processes were. I showed both volunteers how to make SDS-PAGE gels, we made 4 and prepared the 10% APS on our own. 

We got a chance to photograph the gel that I ran on Friday (the one that had been de-staining over the weekend). This worked really well (but note to self don’t make gels symmetrical with the marker in the middle, there is no way to tell which way round it should be, d’oh!).

The gel dried out too much and it caused it to become brittle.

We made more buffers, ones we will be requiring for the western blot at a later date. We made a litre of Transfer buffer and a litre of fresh 10x TBS buffer, which we had to pH using a probe. I never realised how hard this would be, it took forever to get the buffer to the correct pH (7.6) to be exact. The probe was calibrated at the start and washed well between each solution.

We did a streak plate of SUFU using the glycerol stock, we will be using this to make up larger liquid cultures in the future. We made two small liquid cultures of 5ml LB each, 2 using KAN and these were incubated overnight. We prepared 2m 100ml liquid cultures in conical flasks and autoclaved them in preparation for tomorrow.

It was a very productive day, we got through loads of work and it definitely helps having the extra pairs of hands.

30th May 2016 – Day 6

Monday:
Bank holiday extra day at home with my feet up and the family for company! Bonus!

27th May 2016 – Day 5

Last day of the week, cannot believe how quickly that went! There wasn’t too much left to do as Amy wants to leave the big stuff for next week and I am told we will be getting more help, this will definitely be useful always nice to have an extra pair of hands.

The flasks were removed from the overnight incubators, 1ml samples were taken from each. The samples were spun, the supernatant was removed and pellets were frozen (I will need this in a few weeks time). The samples I took yesterday as well as the ones I took today were prepared for another SDS-PAGE gel, they were added to 4x loading dye and pulled through a needle (this was to break open the cells). Gel (which I made on Tuesday and stored in the fridge) was prepared in the tank while the samples were heated at >90˚C for 5 minutes. 20ul of each sample was loaded onto the gel along with 10ul of marker (this is used to tell the size of each separated protein), this was the same process as Tuesday. The gel was stained and then left to de-stain over the 3 day weekend. We will have a look on Tuesday and see if it has worked.

I also used the chilled centrifuge to pellet the reaming cultures in the 50ml falcon tubes, the pellets were then frozen.

The chilled centrifuge: used to pellet the cultures (Clair the technician said it was older than me).

Hard to believe 1st week is finished, its been so exciting. I have loved every minute of getting to know everyone and learn where everything is, whilst getting a taster of what it would actually be like to work in a lab. I loved even loved the hard bits (just means I am being challenged).

26th May 2016 – Day 4

Got my first set of results today! Yay! Today I sorted out the cultures I had set up overnight, and I made glycerol stocks from each LB culture, these get frozen and are used at a later date, while the rest of the overnight cultures got placed in the fridge. I used the bacteria in the fridge and made up 4 larger cultures with antibiotics, these were left to culture until the OD was reached at which point they were induced with IPTG and incubated overnight. (I am good at this now, done it a few times).

….Then I checked the results from yesterday’s SDS-PAGE gel (da da dah….) and it came out well so I’m very pleased. (Go me!!) I photographed the gel using two different machines, these pick up the stain clearly on the protein (I much prefer the machine downstairs, it’s so shinny and new, and much quicker compared-I am sure it’s okay to say I have a favourite).

And for the fun part……!! Giant culture flasks!! These things are massive, I made up 400ml of LB broth in each as well as autoclaved them. I had to use the large autoclave (which takes hours) due to the size of these flasks, I am told we are going to culture the small cultures into these big flasks so we can make lots of protein.

25th May 2016 – Day 3

No sleep last night, my loud neighbours decided to party until 4am …… not great when you plan on getting up at 6.30 am (note to self;  buy earplugs today), going to work tired is not recommended.

Today was busy again, there were a lot of instructions and loads of bits and pieces to do all over the lab. Dr Amy Cherry had some interviews to conduct and gave me a list of instructions and left me to it. They were fairly easy to follow and I managed to finish with plenty of time spare, which I used to chat to some of the technicians about their work and we spoke about what it is like to work in the lab.

Today I took an OD reading followed by a 1ml sample of each (for the gel) from the cultures I had induced overnight. I made an SDS running buffer and then used all of the samples from yesterday and today and ran them on the gels we made up yesterday. 100V then 180V for just over an hour, after which point the gel was stained with Coomassie blue (which turned any proteins visible into a pretty blue colour), then I added de-stain which only removes stain from the non-protein sections of the gel. 

The rest of the cultures were centrifuged and the pellets placed in the freezer (we will use these in a couple of weeks for our western blots). The streak plates that I cultured overnight were removed from the incubator (I always find the LB broth gives off a strange smell, similar to potatoes…always makes me hungry!), a single colony was removed from each and placed into a liquid culture along with antibiotics and this was cultured overnight.

I manged to get a chance to do a little research on PCR primers that I will need for the second half of this study, I have not worked with these primers yet so this was interesting.

24th May 2016 – Day 2

Today was much busier than yesterday, there was a lot to get done and I learnt a lot of new things which was exciting.

The overnight cultures were put through an Axygen-Axyprep Plasmid miniprep spin (kit). It turned out to be a frustrating start to the day as the initial prep kit we wanted to use was empty and so we wasted time fishing around to find a different one.

I prepared larger cultures taken from falcon tubes into conical flasks, these were incubated and the optical density was measured (OD - to find out how many cells are actually floating about, we want them in their log phase where it will be most efficient. Between 0.4 and 0.8). Once required OD was reached, I induced them with IPTG and incubated overnight. I took samples before and after inducing so it could be run on an SDS-Page gel.

Everything I have done up until now I had only learnt about in theory so actually doing it was exciting but at the same time a little daunting. I have run an SDS-Page gel before now, so this at least was not completely new, however before I could actually run the gel, I had to make it……from scratch (normally the technicians prepare these before class, it’s a bit like being on Blue Peter when one magically appears and they say ‘this is one we made earlier’).  I did the resolving first and let it set before I prepared the stacking gel, there are a lot of steps to go through and if one thing goes wrong, it has  to be started again (its not so scary once you get started).

Once this was all completed and the gels were placed in the fridge to store over night, I transformed my cells and these were left to incubate for an hour. I then plated them and left them to incubate overnight (I have done a streak plate before now, but not this many all at once).

Well, day 2 done, there was a lot more waiting around today in between the incubation times which was nice as I got a chance to sit down (but definitely a good call on the trainers…phew!).

First Day!!

First day done!! Started at 9.30 and finished at 18.00, there was a lot to take in and it honestly feels like the longest day in history!! I was introduced to everyone including all of the technicians (those contacts I know will definitely come in handy soon). Not only did I get an introduction to the project and a tour around the labs, I actually got to do a few things myself as well.

I learnt how to use an autoclave and I prepared a few LB broths and LB agar, ready to be used for liquid and streak plate culturing respectively. I also made stock solutions of different antibiotics, ampicillin (AMP) and kanamycin (KAN) (which will be used to select for the correctly transformed bacteria), and a stock solution of IPTG (which is a lactose analogue, it will be used to induce expression of my desired proteins). Each one was filter sterilised near a Bunsen burner and the frozen until we need it. Using what I had prepared, I made a total of 10 different cultures from provided glycerol stocks which I incubated overnight.

Today was an introduction and a chance to get used to the lab whilst preparing some of the things I am going to need over the next few weeks. It was also an introduction to working on my own, which will prepare me for what lies ahead.

I also had the chance to meet some of the third year biochemistry students and listen to their presentations on their dissertations. This was a huge opportunity as well as giving me the chance to ask questions about their studies and gain some insight into their research and about the proteins I will be focusing on over the next few weeks.

Today really opened my eyes to what the next eight weeks is going to be like, but it definitely was a long day (I don’t think my feet have ever hurt so much…note to self: wear more comfortable shoes tomorrow).

Applying for the Studentship!

I applied for the Studentship through the Biochemical Society back in February of this year. The scope of the work is to determine whether there is a correlation between expression of the Hedgehog signalling components and expression of other proteins frequently associated with Acute myeloid leukaemia (AML) and furthermore, whether there is crosstalk between the hedgehog signalling pathway and these other proteins. This is a continuation of Dr Amy Cherry’s previous work and knowledge on Sufu and hedgehog signalling.

The odds of getting a Studentship was extremely unlikely so imagine my surprise but when I received the news in late March that I had been awarded the opportunity…8 weeks of paid glorious lab time… 

On average I have about 4 hours of lab time per week, so not only am I getting additional lab experience, I am putting myself in a position to be better prepared for my dissertation / independent study as well as help prepare me for my PhD ……. excuse me as I just set off a few party poppers (not in the lab!)….. and I will get a taster of what to expect of a career in science.

The only dilemma I have about this studentship is having to write a blog, I am a scientist and it’s not something I am use to, I normally write factual reports (which I still get to do at the end – yay!) but I will sure give it a go.