Another almost purely analysis day. I made my way through ten trials of photobleaching NucS to find the various time intervals each seeable protien took to photobleach. Throwing those into a gigantic vector I used them to help create my experimental curve for the interaction and have ever since been testing different numbers with matlab code for our theoretical curve to make it mimic as much as possible the experimental one. Most of the different variations dont take very long to compile but when I put a very small number in it can lead to quarters of hours disappearing.
This morning I went to a seminar on laser safety which was in french but the slides were in english. I was surprised by how much I was able to follow with this combination. I tended to lose the thread only when someone asked a question either because they spoke too softly, quickly, or perhaps just from the lack of a visual aid.
This afternoon, Rachid and I are doing another set of photobleaching experiments with NucS. We are going to try a different method that he recently read about in a paper to test its effect.
Thursday, June 30
Wednesday, June 29
l'analysis du data
So far today I have already looked at and analyzed two of the datasets from yesterday. Neither one had a single reliable interaction. It takes roughly forty minutes for matlab to perform the analysis after we select our points so I have been spending that time trying to outline the flow for a new matlab program that would make use of the quantum dots to determine the position of the protiens. This is a neccessary step to improve the accuracy of our results and i'm sure would greatly benefit Rachid if i can make it work.
In the meantime Rachid is performing some experiments with NucS, specifically testing the number of nonspecific interactions that still occur even with a surface that has been passivated. So there is no DNA on the surface only our blocking reagent and the streptavidin.
Update: have now completed seven different analyses within one day. The number of reliable interactions are much fewer than we would have liked and a lot of them have too short of a time interval to be sure they interacted at all.
In the meantime Rachid is performing some experiments with NucS, specifically testing the number of nonspecific interactions that still occur even with a surface that has been passivated. So there is no DNA on the surface only our blocking reagent and the streptavidin.
Update: have now completed seven different analyses within one day. The number of reliable interactions are much fewer than we would have liked and a lot of them have too short of a time interval to be sure they interacted at all.
Tuesday, June 28
Just another manic monday (and Tuesday)
So today I did six different trials of the full surface treatment but with different buffers at the end to see if it affected the number of nonspecific protien interactions. Also this morning Rachid and I met again with Cedric and Antigone to talk about the status of the project. It was really interesting because most of the questions that get asked end up leading to basic physics principles and their direct application. For example, Rachid wants to use a different buffer but it would have to be heated to work, so Cedric pointed out that there would be a thermal gradient between the objective lens and the surface. This would induce some convective movements in the thin layer of oil in between the lens and the surface. In order to combat this we would have to make the objective lens the same temperature. Experiments with these types of requirements are normally done inside of a box which is kept at the same temperature always but this poses a problem for us because in order to align the laser it would be necessary to be able to see where it points on the ceiling. Additionally our frequent moving things in and out of the box would also change the temperature.
Then afterwards, Rachid and I went with Antigone to test which wavelengths of light the laser she uses has. We had to use a prism to seperate out the different frequencies and could clearly see a color difference between the various shades of blue and green. Because the laser has also seen many hours of work, two of the weaker frequencies had already stopped working and one of these was the one Antigone wanted.
Yesterday I did some data analysis until my matlab stopped working again :( and also as always assisted Rachid in the Lab with a few trials.
Then afterwards, Rachid and I went with Antigone to test which wavelengths of light the laser she uses has. We had to use a prism to seperate out the different frequencies and could clearly see a color difference between the various shades of blue and green. Because the laser has also seen many hours of work, two of the weaker frequencies had already stopped working and one of these was the one Antigone wanted.
Yesterday I did some data analysis until my matlab stopped working again :( and also as always assisted Rachid in the Lab with a few trials.
Friday, June 24
Lasers and Quasers
actually this post probably won't talk about quasars. See http://en.wikipedia.org/wiki/Quasar instead.
So yesterday we took down the old laser and put up the new old honker of a laser. He works beautifully but is rather on the noisy side as I was forewarned. We aligned all the mirrors and lenses which was really interesting for me since I had never done it before. Then finally after three days of sitting on our hands we did some experiments!
The results didn't come out too snazzy though.
Today, Rachid was working with the students, this is his last class with them, so I began some trials on my own. First I redid the photobleaching experiment. I put NucS on a slide for two minutes, washed it out, put water, and then looked at it with the microscope to find out how long it takes them to photobleac for analysis purposes. For ten of the trials I kept the NucS in water and for another ten I put them in the buffer. Contrary to theory the NucS in water came through a lot better. More were stuck to the surface and were brighter. I redid the entire set of ten trials with buffer after Rachid saw my data but it didn't come through that much better and he said to delete all 20. That's Science I suppose.
Headed to Switzerland this evening to see my Uncle and his family!!!!! I cant describe how excited I am to see them again.
So yesterday we took down the old laser and put up the new old honker of a laser. He works beautifully but is rather on the noisy side as I was forewarned. We aligned all the mirrors and lenses which was really interesting for me since I had never done it before. Then finally after three days of sitting on our hands we did some experiments!
The results didn't come out too snazzy though.
Today, Rachid was working with the students, this is his last class with them, so I began some trials on my own. First I redid the photobleaching experiment. I put NucS on a slide for two minutes, washed it out, put water, and then looked at it with the microscope to find out how long it takes them to photobleac for analysis purposes. For ten of the trials I kept the NucS in water and for another ten I put them in the buffer. Contrary to theory the NucS in water came through a lot better. More were stuck to the surface and were brighter. I redid the entire set of ten trials with buffer after Rachid saw my data but it didn't come through that much better and he said to delete all 20. That's Science I suppose.
Headed to Switzerland this evening to see my Uncle and his family!!!!! I cant describe how excited I am to see them again.
Wednesday, June 22
I can type!
I finally took 2 minutes to try and figure out how to change my keyboard settings so it pretends to be layed out in the American style. It was so easy to fix! Now i'm back to typing at full speed! It was severely handicapping me. The best part is that there's a little switch on the Start Menu bar so anytime Iwant to change back for whatever reason (most likely because I can't remember where a specific symbol is) I just toggle it.
Yesterday there was a RER train workers strike so I had the day off from work. Today I came in hoping against hope that the laser would be checked quickly and we could get to assembling the new one and screwing the mirrors into their new places on the breadboard. However, its now 2pm and the manufacturers still haven't got here. I am still vaguely optimistic that I will do something productive today though.
Initially, this morning I was going to work on the matlab analysis and Rachid reexplained briefly to me some of the steps I needed to take. The second time around everything made more sense and I wrote up my own little matlab-how-to-analyze-your-data guide in my notebook. BUT tragedy struck and the first time through the main routine it ran for almost an hour before we gave up on it. I restarted matlab ready to try again but l'ecole polytechnique has a matlab license that only allows so many people to have permission to run things at any time and in restarting my matlab I lost my place in the queue. So I've been retrying the same command every ten minutes since then hoping some one else will log off matlab and I can begin doing work again. No luck yet though.
Also Cedric and Rachid took a look at the lamp this morning that Cedric uses in his experiments and it was having trouble also! It made me wonder if maybe it wasn't either instrument but actually the power supply in the room or something odd like that. However, Cedric replaced a part on the lamp and now its fine so my momentary hope that nothing was actually wrong with the laser was dashed. At least we can use the lamp if we need to.
Heading to Switzerland this weekend to visit my uncle and his family! Can't wait!
Yesterday there was a RER train workers strike so I had the day off from work. Today I came in hoping against hope that the laser would be checked quickly and we could get to assembling the new one and screwing the mirrors into their new places on the breadboard. However, its now 2pm and the manufacturers still haven't got here. I am still vaguely optimistic that I will do something productive today though.
Initially, this morning I was going to work on the matlab analysis and Rachid reexplained briefly to me some of the steps I needed to take. The second time around everything made more sense and I wrote up my own little matlab-how-to-analyze-your-data guide in my notebook. BUT tragedy struck and the first time through the main routine it ran for almost an hour before we gave up on it. I restarted matlab ready to try again but l'ecole polytechnique has a matlab license that only allows so many people to have permission to run things at any time and in restarting my matlab I lost my place in the queue. So I've been retrying the same command every ten minutes since then hoping some one else will log off matlab and I can begin doing work again. No luck yet though.
Also Cedric and Rachid took a look at the lamp this morning that Cedric uses in his experiments and it was having trouble also! It made me wonder if maybe it wasn't either instrument but actually the power supply in the room or something odd like that. However, Cedric replaced a part on the lamp and now its fine so my momentary hope that nothing was actually wrong with the laser was dashed. At least we can use the lamp if we need to.
Heading to Switzerland this weekend to visit my uncle and his family! Can't wait!
Monday, June 20
Looks like someone's got a case of the Mondays
First piece of news this morning was from Cedric saying that the manufacturer had confirmed that they wouldnt come until wednesday, thus making us lose three days of experimentation which considering that i am only here for 9 weeks is a weighty sum.
Rachid decided we could do at least some rough tests using the UV lamp as our light source instead of the laser. We were also using a DNA that had been marked with a different fluorophore (cy3 instead of alexa) so we needed to change the filters that the light passes through so that only the wavelengths that it absorbs and emits at would pass through. Rachid spent 2 hours screwing and unscrewing different lenses , checking their wavelengths, and testing them with the new DNA. We couldn't see anything. The camera wasnt even working at first but after we rebooted and restarted the program, the DNA still didn't show up. It was clear that the light we saw passing through the lens was not as intense as it should have been but it didn't matter what combination of filters with appropriate ranges we tried, it was still impossible to focus on any plane.
Rachid even tried looking at it optically but had no success. So I have spent the afternoon reading some scientific papers instead. The first one was titled "Visualizing DNA Replication at the Single Molecule Level". It focused on describing the exact step by step instructions for two different methods used to observe DNA replicating itself. The first method bonded beads to the DNA that could then be directly observed with a CCD camera. Depending on how much the beads are calculated to move, the analysis can state how much the DNA has either lengthened or shortened. I am still unclear as to why the DNA would shrink or lengthen and what the correlation between either change would say about the way it replicated itself. The paper was more of a manual so it did not describe the theory behind this behavior or even what their conclusions were after performing the tests. The second method used TIRF! Instead of watching the beads, the growing length of the DNA is observed directly. In both cases flow chambers were used to prepare the surfaces with layers of Blocking Reagent and streptavidin. Their flow chambers consist of three layers, the glass slide, doublesided tape with a channel cut through it, and a slide made from Quartz. The advantage of this method of layer deposition seems to be better control of the uniformity of each layer, but I will have to ask Rachid to be certain.
In reading this paper I focused on where their slide preparation methods deviated from our own as well as where they were similar. Other than the use of flow chambers the other greatest discrepency was that they sonicated their slides to clean them while we use a plasma cleaner. In comparison to their protocol ours is much faster.
The second paper I read was entitled "Single molecule Enzymology" and was largely a discourse on the importance of single molecule studies. The authors put forward that most experiments can only observe the overall average result of an experiment are likely to ignore the small percentage of contradicting results. As a result of this, a lot of information is lost because although the property you are studying maybe something that ought to be uniform to the group it could also be a property of dynamic disorder ( one which fluctuates with time individually for each molecule). To illustrate their claim evidence from one experiment on the enzymatic turnovers of a single cholesterol oxidase molecule were described in depth.
Rachid decided we could do at least some rough tests using the UV lamp as our light source instead of the laser. We were also using a DNA that had been marked with a different fluorophore (cy3 instead of alexa) so we needed to change the filters that the light passes through so that only the wavelengths that it absorbs and emits at would pass through. Rachid spent 2 hours screwing and unscrewing different lenses , checking their wavelengths, and testing them with the new DNA. We couldn't see anything. The camera wasnt even working at first but after we rebooted and restarted the program, the DNA still didn't show up. It was clear that the light we saw passing through the lens was not as intense as it should have been but it didn't matter what combination of filters with appropriate ranges we tried, it was still impossible to focus on any plane.
Rachid even tried looking at it optically but had no success. So I have spent the afternoon reading some scientific papers instead. The first one was titled "Visualizing DNA Replication at the Single Molecule Level". It focused on describing the exact step by step instructions for two different methods used to observe DNA replicating itself. The first method bonded beads to the DNA that could then be directly observed with a CCD camera. Depending on how much the beads are calculated to move, the analysis can state how much the DNA has either lengthened or shortened. I am still unclear as to why the DNA would shrink or lengthen and what the correlation between either change would say about the way it replicated itself. The paper was more of a manual so it did not describe the theory behind this behavior or even what their conclusions were after performing the tests. The second method used TIRF! Instead of watching the beads, the growing length of the DNA is observed directly. In both cases flow chambers were used to prepare the surfaces with layers of Blocking Reagent and streptavidin. Their flow chambers consist of three layers, the glass slide, doublesided tape with a channel cut through it, and a slide made from Quartz. The advantage of this method of layer deposition seems to be better control of the uniformity of each layer, but I will have to ask Rachid to be certain.
In reading this paper I focused on where their slide preparation methods deviated from our own as well as where they were similar. Other than the use of flow chambers the other greatest discrepency was that they sonicated their slides to clean them while we use a plasma cleaner. In comparison to their protocol ours is much faster.
The second paper I read was entitled "Single molecule Enzymology" and was largely a discourse on the importance of single molecule studies. The authors put forward that most experiments can only observe the overall average result of an experiment are likely to ignore the small percentage of contradicting results. As a result of this, a lot of information is lost because although the property you are studying maybe something that ought to be uniform to the group it could also be a property of dynamic disorder ( one which fluctuates with time individually for each molecule). To illustrate their claim evidence from one experiment on the enzymatic turnovers of a single cholesterol oxidase molecule were described in depth.
Saturday, June 18
Playing Catch up
Social Side of Work:
On the bad side of social things, we played our third soccer game this week for the semi-finals and lost 1-0. Which means we're out of the championships. Everyone took it really well though and is talking about next spring already.
Work Side of Work:
Rachid and I were doing experiments trying to do a layer of blocking reagent and then a layer of quantum dots with only 4 to 6 showing at any time. We were having a lot of trouble getting the ratios of each right, and I suggested we put the Quantum Dots on first and figure out what concentration or time we need to only see four-six and then put the blocking reagent to block off the protiens. Rachid LOVED the idea. He pointed out that it'd give us so much more control over the experiment and would be easier to be consistent. He kept saying, "Yes, Yes, that would be good, we're going to do that. Thank you!"
The laser broke. Thursday afternoon it would shut itself off every five minutes (generally when I was in the middle of a video, ugh). Then Friday morning it turned on twice and then after that wouldn't come back up. I showed Cedric and he called the manufacturer and they think the tube of Argon Gas has a problem. Its the most expensive part of the laser so they're not thinking about replacing it right now. Instead they're going to pull 0ut the slightly older laser they used to work with, which is in perfect condition. BUT makes two noises, that of a large Bee and a high pitched whistling...I'm bringing advil to work for the next week. On monday or wednesday the manufacturer is sending someone to look at it and make sure the problem is what we think it is. If they confirm our belief than Rachid and I are going to take apart the entire station and put up the other laser, realign all the mirrors, and hopefully start doing experiments by Friday. It'll be good work experience for me but Rachid's face when Cedric told him the news was pure anguish.
Tuesday, June 14
A New Desk! for the week.
So today I performed two different control experiments. In the first one I put quantum dots and DNA on a slide at the same time and waited five minutes before washing it out and adding buffer. I did four trials where in the first two trials I waited five minutes and in the second set of two trials I waited only 2 trials. The intention was to view whether it would be possible to distinguish the DNA from the quantum dots, since we are hoping to use the quantum dots as markers so that our measurements for the distances between proteins and DNA. It was not difficult to identify the quantum dots or to find the proper positioning for the microscope, but I am not positive I saw any DNA at all. The quantum dots are continuously blinking but all at different rates so it is difficult to determine where I see a particle that fades as it photo bleaches and where I just see a quantum dot becoming temporarily dark. I took three videos but the fourth slide (using the second protocol) leaked and had to be thrown out.
My guesses, without talking to Rachid first, for why the DNA was so difficult to spot would be
-The DNA got accidentally photobleached before being viewed by the microscope (it is almost two weeks old)
-The DNA is there but we have to look very closely to notice it next to the bright multitude of quantum dots
-The Quantum dots were put on the slide first so maybe it was more difficult for the DNA to find places on the slide to stick and thus were more likely to be washed out.
Then after lunch, I set up the second experiment. Here, I added Blocking Reagent with Biotin,waited ten minutes, washed it out, added streptavidin, waited ten minutes, washed it out, and viewed it on the microscope. Ideally since neither of these surfaces have fluorescent markers I should have seen nothing. Of course though, dust and dirt also glow some, although they photobleach even faster than DNA. After observing that it was generally dark, I added the protein and focused on the proper surface. This time the objective was to see how good the blocking reagent was. Ideally, the protein would never come too close to the surface because of the Blocking Reagent, or at least never stick there for several seconds. Instead I should have only observed general brownian motion with the proteins coming near the surface and then being "repelled" in the opposite direction. Without analyzing the video closely, it is difficult to measure the time frames in which the protiens appeared but it seemed that they were in general corresponding to theory.
Friday, June 10
I feel 'sciencey' today
So every Friday, Rachid teaches a lab class for high schoolers. i.e. is not available for me to pester with questions or to give me instructions. So we got here a little earlier. I ran off to quickly clean off the slides with the plasma cleaner. When I returned, we did a quick test with quantum dots to make sure everything was working. I found them quickly enough and everything looked fine, so Rachid gave me his ideas for the different trials I might do and left. We decided to test the effects that various incubation times of Streptavidin had on the concentration of DNA. So far we had always been doing ten minutes, but in some papers Rachid had read that a lot of people used Streptavidin with a concentration one tenth that of ours or incubated it for only 1-2 minutes.
First I set up my four slides in their holders and gave them all an equal treatment of blocking reagent; after ten minutes, i washed them out and put on some streptavidin. After five minutes i washed out two of them and gave them DNA. For the other two I waited five more minutes before washing out the streptavidin. The first one I looked at; I couldn't see anything. I was worried that it had leaked oto the microscope thus ruining the index of refraction. So i took it off and checked it for leaks. It was fine but the process rempved any chance of us using it for further experiments. The next one came out fine and I took a video. Then I took the first one of the five minute set. I searched probably for close to 15 minutes trying to see something on the slides andwould on occasion see some of the white dots but they were so faint that i assumed they were dust. I think maybe I accidentaly photobleached this slide at one point before putting on the microscope. Then I went to pick up the fourth one but noticed that the paper underneath it was wet, so since it was leaking I just cleaned it up and started preparing four new slides.
This time I kept Streptavidin on two of them for five minutes, and on the other two for only two minutes. I got one video from each type. One of the other ones leaked, and I didn't have time to do the last one.
I converted all the videos to Tiff, saved them to a USB, and went back to my desk to begin the analysis but imageJ shows only static at the beginning followed by white. Im trying to google the problem now but so far no luck.
Also there is an LOB barbeque on Tuesday!
First I set up my four slides in their holders and gave them all an equal treatment of blocking reagent; after ten minutes, i washed them out and put on some streptavidin. After five minutes i washed out two of them and gave them DNA. For the other two I waited five more minutes before washing out the streptavidin. The first one I looked at; I couldn't see anything. I was worried that it had leaked oto the microscope thus ruining the index of refraction. So i took it off and checked it for leaks. It was fine but the process rempved any chance of us using it for further experiments. The next one came out fine and I took a video. Then I took the first one of the five minute set. I searched probably for close to 15 minutes trying to see something on the slides andwould on occasion see some of the white dots but they were so faint that i assumed they were dust. I think maybe I accidentaly photobleached this slide at one point before putting on the microscope. Then I went to pick up the fourth one but noticed that the paper underneath it was wet, so since it was leaking I just cleaned it up and started preparing four new slides.
This time I kept Streptavidin on two of them for five minutes, and on the other two for only two minutes. I got one video from each type. One of the other ones leaked, and I didn't have time to do the last one.
I converted all the videos to Tiff, saved them to a USB, and went back to my desk to begin the analysis but imageJ shows only static at the beginning followed by white. Im trying to google the problem now but so far no luck.
Also there is an LOB barbeque on Tuesday!
Wednesday, June 8
They attack from three sides you know...
So today i became better friends with Lauren who is working on her phd, is from Spain, lives on cite universitaire, recognized me while we were waiting for the train, and currently has a hurt foot thus obliging me to step in and play soccer. I also bonded a little with another girl who's name i have forgotten and who asked me to come to lunch with her and some of the other young people so they could get to know me. Then when we realized the soccer game was at the same time as the lunch so instead she came for the second half to cheer us on along with another guy whos name may or may not be guy? pronounced gee i think.
The game was a ton of fun and my team won! Each time is required to have at least one girl playing all the time and if the girl makes a goal it counts for two points so they were very eager to pass me the ball and I played center forward. I wish my skills had been up to par, but at least I wasnt too unagressive and I did get to take one shot (which the goalie easily blocked). I also headed one ball and got the ball away from a player once or twice. I was blocking against a guy probably two feet taller for most of the match so he kept heading balls that i couldnt even jump and reach. Still it was a lot of fun and now we are still in the quarterfinals. Tomorrow I've been invited to play again so I must not have been too bad ;)
In terms of lab work..nothing really went very well. One of my solutions leaked onto the microscope lens which is really bad because then you might never get all the quantum dots off it. Rachid helped me clean it and he said he was fairly sure it would be fine. I have gotten a lot better at finding the particles with the microscope and tuning it only minutely. I have also gained confidence in pipetting things, asking people where things are, finding things on my own, and went and cleqned the slides this mornng with the plasma cleaner all by myself. I wear latex gloves almost the whole day and am still not used to the feeling.
Tomorrow our goal is to find the perfect method for putting just the right amount of quantum dots on a slide so we can use them to measure distances more accurately (for when the whole slide moves) without having them overpower our image. In an ideal world we would have one on each corner of our image when we film. So we can reference both the DNAs and Protiens positions to them, which would be more accurate.
Rachid also said if things keep gong well we might work on a simulation for all this but first we need more data.
p.s. Bonus points to anyone who knows the origin of today's blog's title.
The game was a ton of fun and my team won! Each time is required to have at least one girl playing all the time and if the girl makes a goal it counts for two points so they were very eager to pass me the ball and I played center forward. I wish my skills had been up to par, but at least I wasnt too unagressive and I did get to take one shot (which the goalie easily blocked). I also headed one ball and got the ball away from a player once or twice. I was blocking against a guy probably two feet taller for most of the match so he kept heading balls that i couldnt even jump and reach. Still it was a lot of fun and now we are still in the quarterfinals. Tomorrow I've been invited to play again so I must not have been too bad ;)
In terms of lab work..nothing really went very well. One of my solutions leaked onto the microscope lens which is really bad because then you might never get all the quantum dots off it. Rachid helped me clean it and he said he was fairly sure it would be fine. I have gotten a lot better at finding the particles with the microscope and tuning it only minutely. I have also gained confidence in pipetting things, asking people where things are, finding things on my own, and went and cleqned the slides this mornng with the plasma cleaner all by myself. I wear latex gloves almost the whole day and am still not used to the feeling.
Tomorrow our goal is to find the perfect method for putting just the right amount of quantum dots on a slide so we can use them to measure distances more accurately (for when the whole slide moves) without having them overpower our image. In an ideal world we would have one on each corner of our image when we film. So we can reference both the DNAs and Protiens positions to them, which would be more accurate.
Rachid also said if things keep gong well we might work on a simulation for all this but first we need more data.
p.s. Bonus points to anyone who knows the origin of today's blog's title.
Tuesday, June 7
That was unexpected
So there is a soccer tournament between all the labs at l'ecole polytechnique and they are required to have at least one girl on the team. the girl that usually plays cant tomorrow so i have been recruited. I hqvent played soccer competitively since middle school so this should be interesting...Still i am very excited to be included and i hope to make friends with some more people here :)
1 is the lonliest number
So today I am working on analyzing the seven different sets of data we took yesterday. I have finished the first two and am waiting for the third data set to finish compiling. Basically what I do is open up one of the videos we made of the dna/protien interactions and skim through it to find the clearest picture of the DNA. Then i use this matlab program called suivi that Rachid wrote. It takes the input parameters of the screenshot the dna is on as well as the total number of frames Then it opens up a picture of the frame with the dna andyou pick all the points you think are actually dna and not just dust. You can distinguish between the two because dna is brighter and lasts longer. So i click through the frames of the video and make sure that the points i am selecting last longer than 1 or 2 seconds. I normally pick around 30 points depending on the quality of the trial.
The program goes through each frame of the film,(generally around 2500) searching for high intensity points in the vicinity of the xy coordinates of the dna you picked. high intensit points hopefully correspond to proteins interacting with the dna but are often something else. Then the program generates a massive matrix with all the concievable bits of information you could want about every dna/protein interaction. It also makes a graph for each dna point you selected graphing intensity vs time.
I look through each of these graphs individually. On each two lines are plotted, one corresponding to a large area around the dna points, and another that is nestled closer and thus is generally mpre accurate. I search for spikes in these graphs which would indicate that something bright was right on top of my dna point. for each spike i check the xy coordinates of the bright spot and its distance from the initial dna. If the xy coordinates are rounded to a number ending with .5000 then the point is not accurate and should be thrown out. If the distance fro, the dna is greater than 1.2, the point is also thrown out because the error would be too large. For any points that do make the cut, I keep trackof the x,y coordinates,distance from, the dna, and the number of slides its visible for. This last number is the most important because the average lifetime will indicate what mechanism the protiens are most likely using to repair the dna. Right now we tend to average1-4 good interactions for every 30 dna we track for 2500 slides. This is obviously less than optimal and so we are going to try to improve this statistic by changing how we prepare the solutions (i.e. editing the incubation periods and wash outnumber of times)
Today I met Arnaud and Josephine who are also both interning here but for much longer than me. I went to lunch with the, and theyre a hilarious bunch. I spoke in french as much as i could and they alternated between english and french so i got to practice speaking and understanding french; oh happiest of days!
The program goes through each frame of the film,(generally around 2500) searching for high intensity points in the vicinity of the xy coordinates of the dna you picked. high intensit points hopefully correspond to proteins interacting with the dna but are often something else. Then the program generates a massive matrix with all the concievable bits of information you could want about every dna/protein interaction. It also makes a graph for each dna point you selected graphing intensity vs time.
I look through each of these graphs individually. On each two lines are plotted, one corresponding to a large area around the dna points, and another that is nestled closer and thus is generally mpre accurate. I search for spikes in these graphs which would indicate that something bright was right on top of my dna point. for each spike i check the xy coordinates of the bright spot and its distance from the initial dna. If the xy coordinates are rounded to a number ending with .5000 then the point is not accurate and should be thrown out. If the distance fro, the dna is greater than 1.2, the point is also thrown out because the error would be too large. For any points that do make the cut, I keep trackof the x,y coordinates,distance from, the dna, and the number of slides its visible for. This last number is the most important because the average lifetime will indicate what mechanism the protiens are most likely using to repair the dna. Right now we tend to average1-4 good interactions for every 30 dna we track for 2500 slides. This is obviously less than optimal and so we are going to try to improve this statistic by changing how we prepare the solutions (i.e. editing the incubation periods and wash outnumber of times)
Today I met Arnaud and Josephine who are also both interning here but for much longer than me. I went to lunch with the, and theyre a hilarious bunch. I spoke in french as much as i could and they alternated between english and french so i got to practice speaking and understanding french; oh happiest of days!
Monday, June 6
Science Prevails!
So today, Rachid let me do just about everything by myself! We did seven trials. Six of Protocol 1 and one of Protocol 2. In protocol 1, we wait ten minutes between adding each layer of Blocking Reagent, Streptavidin, and DNA. Each gets washed out once except the DNA which gets washed out three times. In protocol 2, we wait twenty minutes between each layer but the wash out times remain the same. We were supposed to have two trials with protocol 2 but the second one broke and we didn't have any slides left prepared and we have to walk all the way to another building to clean new ones.
For protocol 1 we saw great results and i got to practice finding the DNA with the microscope which is I think the trickiest part of my project so far. I also got to flash the DNA to hasten the photobleaching and then add the protein. I started out as a pretty bad protein-adder. Its important to not touch the liquid on the slide when adding the protein because then you move everything around and your whole analysis will be garbage. Instead you want to slowly drop the protein droplets onto the liquid dna solution.
It was great getting to do almost everything myself with Rachid always asking, What do we do next? or What did we forget? and getting most of the questions right.
On the bad side of things, Rachid said my ID card probably won't be available for another week! Which means I can't let myself in or OUT of buildings for another week. I always need someone with me. bleh. Its a bit of a problem. I can't even get to lunch and back on my own unless i just hang out by the door until someone opens it. stupid.
Tomorrow Rachid is not coming to work because he has to attend a seminar in Paris so I will be spending the day doing analysis on the six different trials we did today or as many of them as I can. and if by some miracle I do finish them all I think i'm going to take a look at the matlab codes that we use. Rachid said if i could optimize them or improve them or add anything to them then thats totally my perogative. Its too bad i dont have matlab on my laptop or i could look at them in the evenings too.
This past weekend we went to the Centre pompidou, Louvre, Notre dame, sat at a cafe with a coffee, and walked all over Paris. This next weekend we're going to Versailles and Marseilles.
Wednesday, June 1
When you hit the wall...climb over it!
So as promised, today I will write a list of some of the things that can go wrong and that have forced us to restart experiments so far. This will probably include some of the innate errors as well.
The biggest obstacle to everything is dust or just not keep things clean enough. In the mornings we go prepare 8-10 circular slides. We take them over to this entirely different building and rinse them with acetone and ethanol. Then we put them in a plasma cleaner. Before producing the plasma we create a vacuum in the chamber and after waiting a minute for all the air in it to disappear we turn the vacuum off and turn on the solenoids which produce a plasma that you can actually see! I will try to get a picture of it before the summer is over but basically it looks like a purple mist. The plasma is ionized and so all the particles on the slides are attracted and pulled off. This is the most effective method to clean the slides. Some other scientists use what is called a hypersonic bath. Basically this entails putting the slides in a solution that is then vibrated at a hypersonic frequency. The drawback to this method is it can only shake off particles that are larger than its wavelength. After the slides are done we put them in a special box Rachid made that we also clean with pressurized air. And then walk back to our building. Now its important whenever we use them to only touch them on the edges and only with tweezers, which can be frustrating when youre trying to align them properly. There is always some dust on them and this is visible and bright in the video although the lifetime of its brightness is significantly shorter than that of the DNA.
Contamination can also come from our pipettes if we are not careful to use a new tip for each new solution we use. Also my hands aren't the steadiest so whenever the tip of the pipette touches the outer rim of the test tubes instead of going straight in to the solution, I also have to change the pipette tip. The same is true for the tip of the mini vacuum we use to "wash out" our slides. Everything that comes in contact with something biological (the tips, the circular slides, regular slides, and our latex gloves) all get thrown away after being used. The only exception are the metal circle things we lock the circle slide in to. THese we rinse well with ethanol and then let dry. We only have four so we can only prepare four tests at one time.
Light is almost as much of an enemy as dust. The fluorescent molecules on the DNA and the protein have to be kept in the dark as much as possible to prevent their being photo-bleached. This means we keep them wrapped p in aluminum foil and tight in our hands when we are getting them out. We also work in the dark with just one side lamp.
Using or creating a bad concentration of something. If there arent enough DNA for instance? We spent the first day just playing around with concentrations.
Once we had a bubble of air in the oil on the lens so we had to clean off the whole lens with this special liquid and special lens cleaning paper. The lens is 10,000 euros-ish so its important to be very careful so that we dont scratch it.
And then of course you can always break something. Like a circular lens for instance which I did yesterday and Rachid did today. They're absurdly fragile.
Today was similar to yesterday except I got to do some more of the stuff on my own, so I think Rachid's trust in me is increasing!
Tomorrow...a fun blog about the plans for this holiday, four day weekend!
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