A lot of things have happened since my last post, including the end of my placement on 19th August. I was so busy with leaving the UK and getting ready for my next year at Max-Planck-Institute in Dresden that I couldn't find time to write anything.
I decided to sum everything up today, when I've just submitted my report to Biochemical Society. The studentship was a fantastic experience and I would recommend it to anybody interested in developing their scientific career. I would personally do it again if I could.
I hope you enjoyed reading about my summer adventure with neurons and fibroblasts. You would be able to read my report and hopefully see some photos on the Biochemical Society website in a while.
Sunday, 4 September 2011
Thursday, 11 August 2011
Towards the end of the placement
It's been almost a week since I last wrote and now my placement is heading towards its finish next week.
This week I finalised cloning the plasmid with mutated HDAC5 gene that I ligated onto pCDNA backbone. I got around 15 colonies of bacteria that grew on agar plates with ampicillin, so I picked 10 of them and extracted DNA using MiniPrep kit. Then I had to check if what I isolated was exactly the plasmid with the mutant gene, so I digested it with a restriction enzyme together with wild-type plasmid to look for an additional 0.7kb piece of DNA, specific to mutant-carrying plasmids. I got this picture:
which shows that 4 of my extracted plasmids (to the left) produce a small piece after the restriction, which is absent in wild type (second right). On the right there is DNA Ladder.
I also carried out my last transfections on Tuesday, this time using a new delivery system, FuGENE, which turns out to really enhance the transfection efficiency. I'm going to stimulate the fibroblasts tomorrow and do my last live imaging on Monday. Now I'm in charge of another live imaging, transfected a week ago by Sangeeta using FuGENE and it looks really promising.
I also quantified another staining of Becky's experiment and it is a bit confusing.
To finish with an extra piece of experience, today I've started my first ever Western Blot, using old Sangeeta's neuronal extracts to test phospho-HDAC antibody. I've started a polyacrylamide gel, I prepared a transfer buffer (on my own) and I'm waiting before I start blotting.
Meanwhile I filled up MiliQ water container and took some tubes and bottles for autoclaving. It will be a bit sad to leave the lab where I feel well settled in.
Enough for now, I'm going to take another picture of my live imaging!
This week I finalised cloning the plasmid with mutated HDAC5 gene that I ligated onto pCDNA backbone. I got around 15 colonies of bacteria that grew on agar plates with ampicillin, so I picked 10 of them and extracted DNA using MiniPrep kit. Then I had to check if what I isolated was exactly the plasmid with the mutant gene, so I digested it with a restriction enzyme together with wild-type plasmid to look for an additional 0.7kb piece of DNA, specific to mutant-carrying plasmids. I got this picture:
which shows that 4 of my extracted plasmids (to the left) produce a small piece after the restriction, which is absent in wild type (second right). On the right there is DNA Ladder.
I also carried out my last transfections on Tuesday, this time using a new delivery system, FuGENE, which turns out to really enhance the transfection efficiency. I'm going to stimulate the fibroblasts tomorrow and do my last live imaging on Monday. Now I'm in charge of another live imaging, transfected a week ago by Sangeeta using FuGENE and it looks really promising.
I also quantified another staining of Becky's experiment and it is a bit confusing.
To finish with an extra piece of experience, today I've started my first ever Western Blot, using old Sangeeta's neuronal extracts to test phospho-HDAC antibody. I've started a polyacrylamide gel, I prepared a transfer buffer (on my own) and I'm waiting before I start blotting.
Meanwhile I filled up MiliQ water container and took some tubes and bottles for autoclaving. It will be a bit sad to leave the lab where I feel well settled in.
Enough for now, I'm going to take another picture of my live imaging!
Friday, 5 August 2011
The best week so far
The fifth week of my placement has just finished. It was really a good week.
My immunocytochemistry of Becky's experiment revealed some very interesting results. For the first time I also quantified my results (I learned how important it was during Cellular & Molecular Imaging module last term) by counting and grouping cells from the images I'd taken. Sangeeta thinks she might publish these results, so maybe I could even get into a publication!
Second, we finally managed to get nice images from live imaging, without microscope breaking or excessive cell death.
When it comes to laboratory practice, I also created a new plasmid with mutant HDAC protein and transfected bacteria with it. And I have a few colonies! I will isolate the plasmid next week and hopefully transfect some fibroblasts with it.
This week was very rewarding. I brought some of the experiments until the end. I didn't start many new ones, but having already 3 experiments in the incubator is really a lot! I keep passaging fibroblasts in my flask so that they're ready for new transfections next week.
My immunocytochemistry of Becky's experiment revealed some very interesting results. For the first time I also quantified my results (I learned how important it was during Cellular & Molecular Imaging module last term) by counting and grouping cells from the images I'd taken. Sangeeta thinks she might publish these results, so maybe I could even get into a publication!
Second, we finally managed to get nice images from live imaging, without microscope breaking or excessive cell death.
When it comes to laboratory practice, I also created a new plasmid with mutant HDAC protein and transfected bacteria with it. And I have a few colonies! I will isolate the plasmid next week and hopefully transfect some fibroblasts with it.
This week was very rewarding. I brought some of the experiments until the end. I didn't start many new ones, but having already 3 experiments in the incubator is really a lot! I keep passaging fibroblasts in my flask so that they're ready for new transfections next week.
Tuesday, 2 August 2011
Promising results - finally?
I haven't written since Thursday, so I'll try to summarise the last few days.
On Friday I had quite a lot to do, but managed to efficiently organise my work. The observations of last immunocytochemistry of Becky's time lapse experiment brought promising results, so we decided to repeat the staining and stain nuclei with Hoechst dye after the weekend. I also stimulated my fibroblasts and fixed them for staining on Monday. I changed media of my other experimental cells and passaged the flask with my fibroblasts. At the end I cut the piece of DNA out of the agarose gel, so that I could extract it later and create mutants.
On Monday I completed the Becky's immunostaing, treated my stimulated fibroblasts with primary antibodies and set up live imaging (again). I also changed media, passaged cells and did quite a few "housekeeping" tasks, especially to do with shuttling between the lab and autoclave room. I like it, though. It makes me feel responsible for the laboratory I'm working in.
Today seems to finally have brought some nice images. My overnight live time course imaging looks interesting, but it will need a thorough analysis before drawing any firm conclusions. However, I also spent almost an hour looking at Becky's stained slides and they look REALLY interesting! I'm looking at the images now and putting them in order and there is a suggestion of a nice pattern.
I already finished staining my stimulated fibroblasts and put them on slides. I'll probably observe them tomorrow. If there's anything else for me to do today, it'll probably be extracting DNA from the gel. But I'm not sure yet.
On Friday I had quite a lot to do, but managed to efficiently organise my work. The observations of last immunocytochemistry of Becky's time lapse experiment brought promising results, so we decided to repeat the staining and stain nuclei with Hoechst dye after the weekend. I also stimulated my fibroblasts and fixed them for staining on Monday. I changed media of my other experimental cells and passaged the flask with my fibroblasts. At the end I cut the piece of DNA out of the agarose gel, so that I could extract it later and create mutants.
On Monday I completed the Becky's immunostaing, treated my stimulated fibroblasts with primary antibodies and set up live imaging (again). I also changed media, passaged cells and did quite a few "housekeeping" tasks, especially to do with shuttling between the lab and autoclave room. I like it, though. It makes me feel responsible for the laboratory I'm working in.
Today seems to finally have brought some nice images. My overnight live time course imaging looks interesting, but it will need a thorough analysis before drawing any firm conclusions. However, I also spent almost an hour looking at Becky's stained slides and they look REALLY interesting! I'm looking at the images now and putting them in order and there is a suggestion of a nice pattern.
I already finished staining my stimulated fibroblasts and put them on slides. I'll probably observe them tomorrow. If there's anything else for me to do today, it'll probably be extracting DNA from the gel. But I'm not sure yet.
Thursday, 28 July 2011
After confocal microscopy
The results that we got after the confocal imaging aren't great, but now I have something nice for presentation and drawing some conclusions. And I know that the most important part of my short summer placement is not getting tons of groundbreaking results, but gaining useful experience. I already feel much more experienced.
Today is rather a lighter day in comparison to previous 3 days. I have 3 experiments in the cell incubator, so there's no rush for new transfections. I'm going to image Becky's cells that I've stained and I'm also going to run my enzyme-digested DNA on an agarose gel for isolation of specific DNA to create the mutant we're interested in expressing in the fibroblasts. Because yesterday the test gel showed that the digestion worked!
Today is rather a lighter day in comparison to previous 3 days. I have 3 experiments in the cell incubator, so there's no rush for new transfections. I'm going to image Becky's cells that I've stained and I'm also going to run my enzyme-digested DNA on an agarose gel for isolation of specific DNA to create the mutant we're interested in expressing in the fibroblasts. Because yesterday the test gel showed that the digestion worked!
Tuesday, 26 July 2011
MiniPrep, Transfections and immunocytochemistry
Now we have a lot of healthy fibroblasts to work with, so I actually do transfections every other day. I transfected some today with fresh DNA that we extracted today in the morning from bacteria.
Unfortunately the cells that I imaged overnight died and I would still need to do it again to get any meaningful results.
I also started another immunocytochemistry of Becky's time course experiment with another antibody.
Tomorrow I will finish the immunostaining, transfect some coverslips with fibroblasts, probably passage another flask of fibroblasts with preparation for another transfection, maybe do some digestions with enzymes and, most importantly, we're going with Sangeeta to analyse my first experiments (still on neurons!) under confocal microscope. I hope to get some results finally!
Unfortunately the cells that I imaged overnight died and I would still need to do it again to get any meaningful results.
I also started another immunocytochemistry of Becky's time course experiment with another antibody.
Tomorrow I will finish the immunostaining, transfect some coverslips with fibroblasts, probably passage another flask of fibroblasts with preparation for another transfection, maybe do some digestions with enzymes and, most importantly, we're going with Sangeeta to analyse my first experiments (still on neurons!) under confocal microscope. I hope to get some results finally!
Monday, 25 July 2011
Working Saturday and next busy week
Since I last wrote on Thursday a few things happened. Our bacteria grew nicely so we'll be able to isolate plasmid DNA tomorrow or today. Unfortunately all the new neurons died, so I won't work on them anymore. However, I started a new experiment on fibroblasts, for which I came to the lab on Saturday to transfect them. I had to come twice and in the morning Zosia came with me and in the afternoon I brought my fiancee Karolina (for safety reasons, people can't come on their own outside working hours, just like I accompanied Becky in my first days - I knew I'd be in that position at some point!).
I just discussed the plan for the week with Sangeeta and it seems to be quite a lot to do. We have a session on confocal microscope booked on Wednesday and I'm looking forward to it. I also just looked at some of Becky's time lapse (the one that involved coming at night) that I stained before the weekend and the staining is nice but we would still like to know more.
I'm now going to observe live cells that I transfected on Saturday.
I just discussed the plan for the week with Sangeeta and it seems to be quite a lot to do. We have a session on confocal microscope booked on Wednesday and I'm looking forward to it. I also just looked at some of Becky's time lapse (the one that involved coming at night) that I stained before the weekend and the staining is nice but we would still like to know more.
I'm now going to observe live cells that I transfected on Saturday.
Thursday, 21 July 2011
Getting bacteria involved
Today me and Zosia got a plasmid each to transform competent bacteria with. We would need them later for transfections. I really liked it because microbial cultures are something I had done quite a lot so I felt confident. For the whole transformation it was mainly following a protocol, but I still did something else.
Neurons and fibroblasts weren't dense enough for passaging today yet, so I will split them tomorrow and transfect on Saturday.
I am also in charge of a live imaging. I have been taking pictures of live cells every two hours since 12.15pm and in 30min I will set the microscope on automatic imaging. Hopefully it won't break this time, with a new light bulb!
Neurons and fibroblasts weren't dense enough for passaging today yet, so I will split them tomorrow and transfect on Saturday.
I am also in charge of a live imaging. I have been taking pictures of live cells every two hours since 12.15pm and in 30min I will set the microscope on automatic imaging. Hopefully it won't break this time, with a new light bulb!
Waiting for new cells
My time lapse experiment started on Monday gave some promising results. However, due to microscope failure I wasn't able to carry on for more than 16 hours and the cells suffered from photobleaching. But now I have a movie on my computer which looks nice and I have my first own documentation of my work. Also, the light bulb of the microscope got changed yesterday, so it should work fine from now on. I'm starting another time lapse observation in a few minutes.
Because of the weekend pH shock on the cells in the incubator, for the past few days we needed new cells. We got some fibroblasts yesterday but today we're getting neurons. Meanwhile I did a few immunocytochemistries (including testing a new antibody), coated new coverslips with polylysine to get them ready for new cells and I also did DNA digestion with restriction enzymes with an aim to create a double mutant protein with Flag epitope tag. I ran test digestion on electrophoresis gel and I have another nice picture for my lab notebook. However, the products are not perfect and some more test digests might be necessary.
Because of the weekend pH shock on the cells in the incubator, for the past few days we needed new cells. We got some fibroblasts yesterday but today we're getting neurons. Meanwhile I did a few immunocytochemistries (including testing a new antibody), coated new coverslips with polylysine to get them ready for new cells and I also did DNA digestion with restriction enzymes with an aim to create a double mutant protein with Flag epitope tag. I ran test digestion on electrophoresis gel and I have another nice picture for my lab notebook. However, the products are not perfect and some more test digests might be necessary.
Monday, 18 July 2011
The promising beginning of the third week
Today wasn't as busy as I expected. It is because of an accidental drop in CO2 concentration in the incubator during the weekend, so the cells that were meant to be transfected didn't survive. Also, the antibodies for immunocytochemistry haven't arrived yet.
However, I set up live imaging of my experiment that started 10 days ago and after 4 hours there are some promising observations! I left it on an automatic mode overnight and hopefully tomorrow I will have the first promising set of results!
Also Zosia came today. She will work together with me on the fibroblasts and neurons for a few days and then she is going to experiment on flies. But it's still nice to have company in the lab. She's staying until after I leave, so I'll have company until the end of my placement.
However, I set up live imaging of my experiment that started 10 days ago and after 4 hours there are some promising observations! I left it on an automatic mode overnight and hopefully tomorrow I will have the first promising set of results!
Also Zosia came today. She will work together with me on the fibroblasts and neurons for a few days and then she is going to experiment on flies. But it's still nice to have company in the lab. She's staying until after I leave, so I'll have company until the end of my placement.
Friday, 15 July 2011
The end of the second week
Today there wasn't really much to do concerning experiments. Two cultures are ready to be stained on Monday and I passaged another flask of cells to make them ready for Monday transfections. I also forgot to mention that I prepared new coverslips for cell growth yesterday by coating them. Today I put cells on them and they should be ready on Monday for a new experiment.
Today was quite calm, but Monday is going to be hectic. Therefore I did a few housekeeping tasks, like collecting PBS stock from supplies office and taking a new bottle for autoclaving, to avoid doing them on Monday. There will be transfections and immunostainings to do. Fortunately, there's another Polish student girl, Zofia, coming for a month from Monday. It will be fun to have extra company, especially from the same country!
Today was quite calm, but Monday is going to be hectic. Therefore I did a few housekeeping tasks, like collecting PBS stock from supplies office and taking a new bottle for autoclaving, to avoid doing them on Monday. There will be transfections and immunostainings to do. Fortunately, there's another Polish student girl, Zofia, coming for a month from Monday. It will be fun to have extra company, especially from the same country!
Towards the end of the second week
Unfortunately my observations of live cells didn't give much results. Throughout 5 hours hardly anything changed, so the fluorescent dots under investigation might not have even been live cells. Sad, but life and science go on, I have another set of cells to observe.
Yesterday we also looked at the first analysis of Becky's experiment, in which I accompanied her at nights. The first staining didn't show as much as we expected - the signal didn't shuttle as we hoped, but at least we found a way of stimulation. There is still more cells for investigation.
Today I'm carrying on with the last of my experiments started on Monday. I'm also passaging cells and getting them ready for new transfections next week. Next week new neurons will be available, so I'll come back to working with them rather than fibroblasts.
I also realised that posting before I start work helps me think about what I'm meant to do on the day and focus. Losing internet in the house wasn't that bad!
Yesterday we also looked at the first analysis of Becky's experiment, in which I accompanied her at nights. The first staining didn't show as much as we expected - the signal didn't shuttle as we hoped, but at least we found a way of stimulation. There is still more cells for investigation.
Today I'm carrying on with the last of my experiments started on Monday. I'm also passaging cells and getting them ready for new transfections next week. Next week new neurons will be available, so I'll come back to working with them rather than fibroblasts.
I also realised that posting before I start work helps me think about what I'm meant to do on the day and focus. Losing internet in the house wasn't that bad!
Thursday, 14 July 2011
Looking at life
Today I'm in charge of my first own time-course experiment. Every hour I look at living fibroblasts in a dish (on a specific medium) and hope for some changes between images. At the end I should have a sequence from which a short movie could be produced, using ImageJ software, which I already downloaded on my laptop and had already used during Cellular & Molecular Imaging module practicals.
In between taking images I finished immunostaining of Sangeeta's experiment and made a microscope slide, threw away dying cells from cultures, looked at other cells if they are happy or need passaging and cleaned the desk with ethanol. The lab is my workplace and I've found myself trying to keep it as tidy as possible. I think it should be helpful when I start working in a bit more crowded laboratory (in honesty - working in a less crowded one wouldn't be possible, but I like it!). I see good points of my summer practice every day.
In between taking images I finished immunostaining of Sangeeta's experiment and made a microscope slide, threw away dying cells from cultures, looked at other cells if they are happy or need passaging and cleaned the desk with ethanol. The lab is my workplace and I've found myself trying to keep it as tidy as possible. I think it should be helpful when I start working in a bit more crowded laboratory (in honesty - working in a less crowded one wouldn't be possible, but I like it!). I see good points of my summer practice every day.
Waiting for results
Yesterday I looked at my new slides after transfections and immunocytochemistry under the microscope. Transfections don't work perfectly and aren't very reliable. Antibodies provide much better signal. Consequently, I can't draw too many conclusions from my experiments yet. However, through trying different approaches we should be able to optimise the experimental conditions for the most important experiments. Step by step, I'm getting closer to the experiment that should answer the main question of my project.
While working on my experiments, I do various things in which I get more and more confidence and independence. I improved in handling coverslips and passaging the cells. I can also moreless assess under light microscope if the cells need passaging. I can also switch on the fluorescent microscope without Sangeeta's help. It's not an easy task!
Today I might have to do a few things on my own, because Sangeeta will have to attend meetings. I'm quite curious what it'd be like.
While working on my experiments, I do various things in which I get more and more confidence and independence. I improved in handling coverslips and passaging the cells. I can also moreless assess under light microscope if the cells need passaging. I can also switch on the fluorescent microscope without Sangeeta's help. It's not an easy task!
Today I might have to do a few things on my own, because Sangeeta will have to attend meetings. I'm quite curious what it'd be like.
Wednesday, 13 July 2011
New experiment
After my post yesterday, it turned out the day wasn't over yet. After the lunch break I observed my slide under the microscope, but the transfection with GFP didn't work well. Immunocytochemistry would be necessary to see anything meaningful.
Apart from that, I started a new experiment with a new transfection of fibroblasts that I passaged on Monday. Now I am going to change their medium and treat the cells from previous experiment with secondary antibodies.
I also got my exam results today and I'm really happy!
Apart from that, I started a new experiment with a new transfection of fibroblasts that I passaged on Monday. Now I am going to change their medium and treat the cells from previous experiment with secondary antibodies.
I also got my exam results today and I'm really happy!
Tuesday, 12 July 2011
Everyday working in the lab
I didn't write anything yesterday because the internet access in my house finished for the year and until the end of the placement I will have to use the web on campus. Now I'm writing from the department during my lunch break.
Yesterday and today I carried on with my experiment. I have 4 cell lines now and each of them requires different handling. I have been changing media, preparing starvation medium, stimulating cells, fixing them, immunostaining with antibodies and preparing slides for fluorescent microscopy. There is quite a lot to do now and most importantly - I can do it on my own. I feel confident now, but I still need Sangeeta to show me where necessary antibodies or chemicals are.
Both Monday and Tuesday were similar - we discussed the plan with Sangeeta in the morning and then I did what I had to do. I am really enjoying the work. Also, I clearly understand what we're doing, which makes it really exciting!
Apart from the experiment, I also feel so settled in the lab that I also carry out "housekeeping" duties, such as putting tips in the boxes, shuttling between the lab and autoclave room and filling water bath with deionised water. I'm feeling part of the lab.
I also noticed that the times of my posts on this blog are from American time zone (-8 hours). I usually write in the afternoon, not at 6 in the morning!
Yesterday and today I carried on with my experiment. I have 4 cell lines now and each of them requires different handling. I have been changing media, preparing starvation medium, stimulating cells, fixing them, immunostaining with antibodies and preparing slides for fluorescent microscopy. There is quite a lot to do now and most importantly - I can do it on my own. I feel confident now, but I still need Sangeeta to show me where necessary antibodies or chemicals are.
Both Monday and Tuesday were similar - we discussed the plan with Sangeeta in the morning and then I did what I had to do. I am really enjoying the work. Also, I clearly understand what we're doing, which makes it really exciting!
Apart from the experiment, I also feel so settled in the lab that I also carry out "housekeeping" duties, such as putting tips in the boxes, shuttling between the lab and autoclave room and filling water bath with deionised water. I'm feeling part of the lab.
I also noticed that the times of my posts on this blog are from American time zone (-8 hours). I usually write in the afternoon, not at 6 in the morning!
Friday, 8 July 2011
End of first week
On the last day of the first week I got whole protocols to do on my own. I carried out fibroblast transfections for the beginning of my second experiment, from the beginning until the end (Sangeeta's role was only to give me the plasmid DNA and show where necessary chemicals were, but for the next time I wouldn't even need that much!). Wanting to overcome a few problems, I showed some initiative, which might result in some future problems with the experiment, but hopefully it will work fine. I was also in charge of three last pictures of live fluorescent microscopy and passaging cells at the end of the day. I only learned these techniques yesterday while Sangeeta was doing them! She clearly trusts me. Now I feel much more confident in the lab. I come in the morning, I get the plan and I do it. I'm happy with myself.
An extra bonus for today - I got my first payment, coming from the Biochemical Society. I look forward to the next week.
An extra bonus for today - I got my first payment, coming from the Biochemical Society. I look forward to the next week.
Thursday, 7 July 2011
Gaining independence
After yesterday's demonstrations of immunostaining and slides preparations, I earned enough trust to be allowed to apply the secondary antibodies and mount the cells on a slide completely on my own. I see myself gaining more and more confidence in the techniques I would often need throughout the placement. However, Sangeeta said that if there was any method I would especially like to practise, we can come up with an additional experiment so I can gain extra skills. Which means that I might do my first ever Western blot at some point.
Lab work always involves a lot of waiting, caused by washing, incubations, staining, drying or defrosting. I spend my breaks practising handling coverslips, very fragile and used a lot. So far the breaks have been long enough so that I improved a lot in picking coverslips and even managed to turn them around. Obviously, when there are delicate cells residing on them, there is more pressure, but I must believe that practice makes perfect.
There was also a lot of fluorescent microscopy today. First I observed my results and because of higher magnifcations not working they would probably have to wait for confocal microscopy to poduce better data. However, I have put my first conclusions in my lab notebook.
Afterwards Sangeeta put me in charge of the first part of life imaging of her experiment. I am really gaining independence and self-confidence! I also find my way around the lab and department much easier now than on Monday - clearly I am setlling down. Which sounds good, seeing myself spending there the next six weeks. They should be really interesting and we had a productive discussion about the plans and projects with Sangeeta today.
Tomorrow we are going to move from neurons to fibroblasts. The difference is quite big. Hopefully I will adjust quickly and will be able to fill my notebook with more experiments!
Lab work always involves a lot of waiting, caused by washing, incubations, staining, drying or defrosting. I spend my breaks practising handling coverslips, very fragile and used a lot. So far the breaks have been long enough so that I improved a lot in picking coverslips and even managed to turn them around. Obviously, when there are delicate cells residing on them, there is more pressure, but I must believe that practice makes perfect.
There was also a lot of fluorescent microscopy today. First I observed my results and because of higher magnifcations not working they would probably have to wait for confocal microscopy to poduce better data. However, I have put my first conclusions in my lab notebook.
Afterwards Sangeeta put me in charge of the first part of life imaging of her experiment. I am really gaining independence and self-confidence! I also find my way around the lab and department much easier now than on Monday - clearly I am setlling down. Which sounds good, seeing myself spending there the next six weeks. They should be really interesting and we had a productive discussion about the plans and projects with Sangeeta today.
Tomorrow we are going to move from neurons to fibroblasts. The difference is quite big. Hopefully I will adjust quickly and will be able to fill my notebook with more experiments!
Wednesday, 6 July 2011
Night experience and the first experiment
I spent two nights visiting the lab every 4 hours - at 10pm, 2am and 6am on Monday/Tuesday night and 10pm and 2am on Tuesday/Wednesday, helping with fixing the cells from Becky's time-lapse experiment. It turned out to be really useful, because I actually got to fix the neurons before I had to do the same with the cells for my experiment.
Yesterday I got a day off to read the review and get some rest after the night lab work. Today I started with planning the course of the experiment with Sangeeta. An actual, real experiment, with controls and duplicates. Before starting I got essential practice with handling coverslips in the non-cellular environment, not to risk any losses (it would be really sad to lose the neurons that looked really good under the microscope). After a few trials I got enough confidence. Meanwhile I got familiarised to the kit used for extracting plasmids from bacteria by Becky (which wasn't too bad, as I remembered doing that a few years ago in Warsaw, during my first lab experience).
Then the time came for my first experimental set-up. Everything had to be sterile, under the hood. Despite a few initial struggles with coverslips with cells, I managed to put everything right for incubation, so that after lunch I could fix them, just as I learned at nights.
After fixing I prepared some of the neurons for immunocytochemistry (another theoretical module, Cellular&Molecular Imaging, brought into life!) and Sangeeta showed me how to prepare the slides for fluorescent microscopy. Which meant that on the same day I could still observe the initial outcomes of my first experiment! So far they look promising, but I should know more tomorrow, after confocal microscopy.
Before looking at my slide, I also got introduced to the fluorescent microscope in the labs. I know I'll use it a lot over the next weeks and it doesn't look that scary, operating it shouldn't be a big trouble. However, Sangeeta promised I would get more practice tomorrow morning.
Today was also Becky's last day in the laboratory, so from tomorrow on it's going to be only Sangeeta and me. It means there might not be somebody to lead me through anymore, but on the other hand it means I'll have to get independent and self-confident quicker, which isn't bad at all.
Yesterday I got a day off to read the review and get some rest after the night lab work. Today I started with planning the course of the experiment with Sangeeta. An actual, real experiment, with controls and duplicates. Before starting I got essential practice with handling coverslips in the non-cellular environment, not to risk any losses (it would be really sad to lose the neurons that looked really good under the microscope). After a few trials I got enough confidence. Meanwhile I got familiarised to the kit used for extracting plasmids from bacteria by Becky (which wasn't too bad, as I remembered doing that a few years ago in Warsaw, during my first lab experience).
Then the time came for my first experimental set-up. Everything had to be sterile, under the hood. Despite a few initial struggles with coverslips with cells, I managed to put everything right for incubation, so that after lunch I could fix them, just as I learned at nights.
After fixing I prepared some of the neurons for immunocytochemistry (another theoretical module, Cellular&Molecular Imaging, brought into life!) and Sangeeta showed me how to prepare the slides for fluorescent microscopy. Which meant that on the same day I could still observe the initial outcomes of my first experiment! So far they look promising, but I should know more tomorrow, after confocal microscopy.
Before looking at my slide, I also got introduced to the fluorescent microscope in the labs. I know I'll use it a lot over the next weeks and it doesn't look that scary, operating it shouldn't be a big trouble. However, Sangeeta promised I would get more practice tomorrow morning.
Today was also Becky's last day in the laboratory, so from tomorrow on it's going to be only Sangeeta and me. It means there might not be somebody to lead me through anymore, but on the other hand it means I'll have to get independent and self-confident quicker, which isn't bad at all.
Monday, 4 July 2011
Placement: the beginning
I'm Tomek and last Friday I officially finished my second year of my Bachelor in Genetics at the University of York, taking the last exam of the year on Developmental Biology. Even though the weather in my home country, Poland, is really summer-like now and many of my old friends would now go sunbathing, I decided to stay in York until my student house let expires and gain experience in lab.
Everything started in January when I applied for a summer internship project to Dr Sangeeta Chawla, who works in my Biology Department in York. The idea of working with neurons at the molecular level, including studying transcription factors, was spot on for a future (hopefully!) molecular geneticist. A nice bonus was the funding by the Biochemical Society that I managed to be granted.
Today, Monday 4th July, after one weekend of holidays, was the first day of my summer placement. Sangeeta's group consisted of two people before I came - her and Becky, a final year undergraduate finishing her project, who I have already known for a few months. This really made the first day as good as it could be, because Becky could take me through transfecting neurons step by step, right from the start. After getting my personal lab notebook and getting familiar with H&S rules, I got my own (beautiful!) nervous cells in a dish and transfected them with liposomes - which meant I brought into life the knowledge from the lectures on creating transgenic animals from Applied Genetics module. So there seems to be a point in going to lectures during my degree! Even though I was as fresh to the lab group as Morrison's fruits, I actually did proper labwork from the first day, under the hood, with ethanol disinfecting and careful handling live cells. I even got my own plastic tray for keeping Eppendorfs in the freezer! And I have the first protocol written down in my lab notebook. Definitely not the last one.
The day is not over though. Tonight I'm accompanying Becky with her time course experiment, which means we need to go to the lab at 10pm, 2am and 6am! Excited! In a few weeks, this might happen again, but then I will be the one needing company...
Not to get too bored, I got a review to read from Sangeeta, so that tomorrow, after I get enough sleep after the night visits to the lab, we can thoroughly discuss my experiments. My neurons are now ready, so why keep them waiting? It should all get in the full swing on Wednesday!
I'll try to blog as often as possible, to share all my feelings about the placement and keep the updates on my progress. I'm sure the next posts won't be that huge, I just wanted a bit of introduction here. Hopefully you won't get bored!
Tomek
Everything started in January when I applied for a summer internship project to Dr Sangeeta Chawla, who works in my Biology Department in York. The idea of working with neurons at the molecular level, including studying transcription factors, was spot on for a future (hopefully!) molecular geneticist. A nice bonus was the funding by the Biochemical Society that I managed to be granted.
Today, Monday 4th July, after one weekend of holidays, was the first day of my summer placement. Sangeeta's group consisted of two people before I came - her and Becky, a final year undergraduate finishing her project, who I have already known for a few months. This really made the first day as good as it could be, because Becky could take me through transfecting neurons step by step, right from the start. After getting my personal lab notebook and getting familiar with H&S rules, I got my own (beautiful!) nervous cells in a dish and transfected them with liposomes - which meant I brought into life the knowledge from the lectures on creating transgenic animals from Applied Genetics module. So there seems to be a point in going to lectures during my degree! Even though I was as fresh to the lab group as Morrison's fruits, I actually did proper labwork from the first day, under the hood, with ethanol disinfecting and careful handling live cells. I even got my own plastic tray for keeping Eppendorfs in the freezer! And I have the first protocol written down in my lab notebook. Definitely not the last one.
The day is not over though. Tonight I'm accompanying Becky with her time course experiment, which means we need to go to the lab at 10pm, 2am and 6am! Excited! In a few weeks, this might happen again, but then I will be the one needing company...
Not to get too bored, I got a review to read from Sangeeta, so that tomorrow, after I get enough sleep after the night visits to the lab, we can thoroughly discuss my experiments. My neurons are now ready, so why keep them waiting? It should all get in the full swing on Wednesday!
I'll try to blog as often as possible, to share all my feelings about the placement and keep the updates on my progress. I'm sure the next posts won't be that huge, I just wanted a bit of introduction here. Hopefully you won't get bored!
Tomek
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