Week Three: MONDAY

Today I accomplished a couple of things; reading and code-documenting, not necessarily in that order. I generally like to break up the day as a mix between reading and viewing code, so that neither become to monotonous.

I finished reading Kruithof's thesis, Magnetic Tweezers Based Force Spectroscopy Studies of the Structure and Dynamics of Nucleosomes and Chromatin (2009), which was quite a read; 9 out of 10, would recommend. I also began reading a thesis by Fan-Tso Chien, Chromatin Dynamics Resolved with Force Spectroscopy (2011), which I plan to finish tomorrow. In general these readings have strengthened my understanding of force spectroscopy. More so, it highlighted more things that I should learn about. I've slowly been creating a list of things to delve more deeply into, in no particular order. Since this project is on magnetic tweezers, it would be helpful to further my knowledge on microbiology, so I've included topics such as DNA, chromatin, and the worm-like-chain model, mostly with respect to their use in force spectroscopy experiments, and what questions we can resolve about chromatin through force spectroscopy. I've also included more technical things, some which I know a little about already, and others which I feel I should know but unfortunately don't since I've never had a class in Bayesian statistics. These are, Brownian motion, Monte-Carlo experiments, and Markov analysis w/ respect to a Hidden Markov model.

Back to the mechanical side of things, I made a couple of important breakthroughs, but I'm not entirely sure of their implications. I analyzed how the motor control board and our code for the control board works more thoroughly. When I initially ran the code, I found that the setup thought it was at initial coordinate setting of -16,777,216 (units, still need to figure out what the units are). Not remembering what I had left the motor at yesterday, I assumed this might have been just where I left off, although it was highly unlikely. I told the code to move the motor to a position of 0. After this, I proceeded to unplug the motor control board from power and the computer. I closed out all of the code as well. Reconnecting everything, the position remains at 0. I then told the code to move to 100,000 and verify it was at 100,000. Closing and unplugging everything only to reconnect later, I found the position was now 0. I noticed that after a move to 100,000, the motor was at roughly the same position as 0. To verify that 100,000 wasn't simply a "rotational multiple of 0" (like on the unit circle, 0, 2pi, so forth), I made a move to 50,000, believing that this should be somewhere in half, and give an initialized number. This is not the case however. This will also read 0 on startup, as well as any non-multiple of these. What this means is, the control board does not hold memory after power down. I also noticed that the absolute moves as used in the code, 25,000, 50,000, 100,000, and 700,000 are not multiples of one another, as they do not remain at the same position. What this also means is that there will be a problem with initialization for absolute moves. Any position the motor is in at shutoff will become the new 0 at startup, so absolute moves no longer are absolute. I also have no idea why the code believed the motor was at -16,777,216 at startup.

I found what I thought could be possible Piezo-related subvi's. All of them are contained within the SimpleMove vi, or within the Piezo-related subvi's as additional Piezo sub vi's. This is good, as it means it should be relatively easy to remove the part of the case structure in SimpleMove that contains the code for the Piezo with little repercussions.

Finally, I began exploring the code above SimpleMove. I found that SimpleMove has instances in InitializeLUT, InitTweezers, MoveTrajectory, the main instance of the code, and UseParFile. I began my documentation of MoveTrajectory, although it is quite a complicated bit of code. For tomorrow, I plan to continue to document these vi's. Also, out of curiosity, I will find what the code believes to be the initial position of the motor.    

BioSAXS Controls Video

Video of System Running: The first computer screen shows a spreadsheet of all the runs with information about each run; what it is, how many seconds its exposed for each image, how many images, and the time that the run began. The second computer screen controls the spec to make each run start and to vary the time of the run. This screen also controls the sample cell to tell it to move the sample left or right, to oscillate and at what step size it should take. The TV screen above both shows what the camera sees so that the oscillations can be regulated and we can be sure that no aggregates or bubbles are in the cell. Above all of this is the sensors for I2, I3, GDoor and the diode. The diode does not give us much information. When I2 and I3 are large, that signifies the strength of the X-rays in the beam.

Week Two: BioSAXS

I accompanied Professor Andresen to the Cornell High Energy Synchrotron Source (CHESS) to run SAXS experiments on various nucleosome arrays. Friday and a large part of Saturday, we modified the experimental setup. We elongated the tubing in the hutch, inserted a beam stop, switched the detector from a 100K pixel to a 200K and began with an X-ray of 200 by 200 micrometers. 

Inside the hutch, overall view of the setup. The long tube farthest to the left was exchanged with the tube on the wall behind to lower the q value. The X-ray beam enters at the far right.

Further to the right the 200K detector can be seen which is the box on the end of the tubing. Right before the detector is a glass plate that the beam stop is placed on. **Fun Fact: Professor Andresen, as a graduate student, painted the maroon square behind the detector.

Then a camera was set up to look at the sample inside the beam mostly to ensure that the sample was centered at the beam and to look for possible air bubbles or aggregates in the sample. At this point the guards and elements of the tube were varied to get the best beam signal throughout the tube with minimal scattering due to the sides of the tubes. Silver behenate was used as a calibration of the beam to find rough values for q to see what range we were working with. All of this was changed in the hopes of lowing the q value, which is the momentum transfer or scattering vector, to a low of 0.004. After the physical setup was chosen and implemented, all the different elements had to be synced with the computer outside the hutch (the hutch is the room where the X-rays are present during experiments).This was what took the most time because so many variables were changed since the last time the G1 hutch was operating. 

Operating system outside of hutch including the real time values of I3 and I2 which give an idea of the strength of the X-rays inside the hutch, the camera monitor that is looking at the sample, and all of the programs that control the beam, guards, sample etc. 

In the image above, Professor Andresen was closing the hutch door and turning the key. This is where the key is placed and the X-rays are turned on. After searching the hutch (pressing two buttons on either side of the hutch and ensuring no one is inside) the door is shut, key is taken and placed in the Station G1 control and the black buttons are pressed on the top right corner of the control box. 

 The element that gave the most trouble was the beam stop which was not in the correct place for running initally and also had different components that continually got in the way of the X-ray beam. This caused the I3 signal to be very low (~16) until it was fixed and showed a signal of ~200. After many hours of centering the beam and correcting the scattering from the sides, we began taking data from our samples. 
We began with the Trimer A which is three nucleosomes together because it is the smallest set of nucleosomes we will look at this weekend. First, distilled water is sent through the sample cell (about 3 times), then ethanol is sent through (about 2 times). At this point the sample cell is mostly clean so we turned on the air to dry the area for about 30 seconds minimum. After all that, we inserted a buffer, closed the hutch, and ran the buffer. This gives us a base line to subtract out any systematic problems against the actual samples. 

View of where the sample is inserted. Between the two plates, a small clear sample cell can be seen. This is where the samples, buffers, water and alcohol is placed and also where the air comes out. The camera is on the opposite side of the beam.  

We ran everything for 5 seconds with 20 images with a ten second break and then once more. After the buffer, we cleaned the sample cell with water, alcohol, and air once more and put the sample inside. We repeated the set up of 5 seconds, 20 images twice for the sample. This procedure was followed for all of Trimer A which included three dilutions (1x, 2x, 4x) for buffers of 10, 50, 100 and 200. 
Today, we are running samples of higher nucleosome arrays. Tetramers and dodecamers (4 and 12 nucleosome arrays respectfully) are much larger structures so the set up had to be modified so that the scattering angles could be measured. This is because larger objects give smaller scattering angles and smaller objects give larger scattering angles. We had some issues with a peak coming off in the z direction from the beam but by changing the guards and the beam stop, this was corrected. Because we are working with the other samples, we lower the beam size to 100 micrometers in the z direction which also lower our intensity so we will be taking longer time intervals when we run. 

Week 2: Friday

Today I worked primarily on experimenting with the motor control VI and documenting the SimpleMove vi and its sub vi's. I also did a little bit of reading on magnetic tweezers. But first, I'd like to share with you the current setup we have for the lab.

We took the floater table from the nuclear lab and moved it into the middle of the room. Also featured is the new computer.

I started the day by messing around with the motor control board, the motor, and the VI that's supposed to run it. I improved over my messing with it from the other day by managing to successfully make it rotate in the opposite direction today. Although I initially thought that the code written for the control board would be bug free, I discovered later in the day that there would be certain cases in which the code would break. For example, starting from point zero, it is possible to move the motor in the wrong direction into negative absolute points. This could be bad for the MT setup, as it could result in the motor moving when it cannot physically move due to a barrier. I also found that when just launching the VI, the code does not always recognize what is zero for the motor.

The motor setup: A control board straight out of the box (or in it), a motor, and a breadboard. A USB cable leads to the computer. A power supply runs off of the table to the control board.

A picture of the control board that we are using. It is a Trinamic TMCM 6110 motion control board.

I also ran into issues today with the code that calls the move controls for the motor. Yesterday I attempted to see if deleting parts of that code that don't pertain to the motor control would allow for it to run even though we don't have the other parts that the parts of the code that I deleted refer to. Unfortunately, today I found that it still will not be able to run, as the unit calibration and timing sub vi's will not run due to missing files. It's possible that if this overhead code that calls the motor control code fixes the problems with zeroing I mentioned earlier, but I'm not 100% sure.

Week 2: Thursday

Today I reviewed the new LabVIEW code sent to us. We finally have our new computer up and running in the lab installed with LabVIEW. I managed to make the motor control board operate the motors through the supplier's LabVIEW code just as Dr. Andresen had. However, I found that the existing MT LabVIEW code could also run the motors. While I was quite pleased with this, I found it to be somewhat buggy, and had no way to make the motor move in the opposite direction, which is disappointing. I believe it to be a problem with some uninitialized values, which might be initialized or transformed in some other VI in the codeset.

For the rest of the day, I began looking at the SimpleMove.vi, the part of the code that basically tells the motor control to move. It also does several other things, however, it's not functional right now, since there are several pieces of the code missing. With our optics in order and just waiting to be ordered, it is now my job to figure out how to get this section of the code running again, which should take a couple of days.

Week 2 Wednesday

For the most part this week, I've been figuring out the optical system and components that we are going to include in the magnetic tweezers setup. It's been problematic so far, because we're having difficulty with the parts list from one of Professor Andresen's friends. Complicating the situation is the fact that they're in Singapore, so any communication is delayed significantly.

One of the first problems we have is determining how to focus the collimated light coming out of the objective. This light has to be focused in order to be stored on our CCD. We have a choice of either a f=100 or f=180mm lens. However, we're unsure if (and what would happen) if we include both lenses in the setup at the same time. Secondly, we need to figure out what type of LED we want as our light source. We're leaning to using Thorlabs as our main supplier for all of our opto-mechanical and optics equipment for ease. So, for the most part of this afternoon, I've been chatting with a tech-adviser from Thorlabs for help with selecting the LED and the appropriate opto-mechanical components. Right now we're leaning towards a red (625nm) LED w/ an output power of 1000mA. To collimate the LED, we'll be constructing our own collimator out of different Thorlab stuff. We'll be using an aspherical lens with AR coating in some appropriate housing to collimate the light from the LED. This housing will attach to the LED itself. Of course, the main problem now is making sure that the whole list of everything we need is complete, and get everything ordered.

Week Two: Mike Mike Mike Mike! Guess what day it is!?!

This morning, I reanalyzed the cobalt- only samples on the ICP, exported the raw data, and added the information to the graphs I made yesterday. The fraction of charge neutralized by cobalt was within the standard deviation from the first run but the individual concentrations of cobalt and phosphorus were significantly lower than yesterday. Although the second run had much lower concentrations, the trendline for both ions traced the same as the first run. This afternoon, I have been catching up on my reading about DNA, nucleosomes, SAXS and studies related to my experiment. 

Week Two: Tuesday

I recreated the graphs from yesterday of the fraction of charge neutralized by Cobalt and Sodium among others using data from both of the runs. Below are the separate graphs of the charge neutralized by the ions. 

I also began to run the other samples given to us with no sodium present in large or varying quantities and only cobalt was varied. Tomorrow I will run the samples again to verify the initial results. 

Week Two: Monday

I analyzed the data collected from my final run last week to determine the quality of the run based on the knowledge I have about how the concentrations of cobalt, sodium and phosphorus change with increasing sodium levels. Using the fact that each of the samples increased in sodium from 0 mM to 80 mM, I created Excel graphs to show the relationships between each of the ions and also how the system competitively behaves. I also determined the relative proportions of cobalt and sodium in solution and created a graph to show their congruence relative to the amount of phosphorus detected. This is shown in the graph below. 

The error bars are rather large so I will be adding a second run of the same samples to clean the graphs up. Today, Steve and I also had a group meeting with Prof. Andresen to discuss the current state of our experiments, mostly so each of us understand what the other is doing, and also discussed what we will be doing for the next two weeks. 

Settimana Due Giorno Uno

Oggi io ho letto molti documenti per il mio lavoro. In the morning I read up on optics, particularly relating to ccd cameras and camera focusing in general. A problem that we've encountered is that, while the focus from the back end of the objective in the setup may be infinity, we still need a way to resolve the picture so that it is in focus for the ccd camera. We need a lens, and a couple of other optics to get the setup to work properly.
I also began to document and review parts lists for other magnetic tweezers setups. I will begin comparing these groups' parts lists to our own, and figure out which are necessary.
Around 3.30, the motor control board and the motor itself arrived. I began to play with the motor control board by connecting it to my laptop via usb. A green light came on flashing on the board, which is a good sign. After a little hassle, I believe that I have my laptop using the right driver for the control board. Hopefully my pc will now recognize the device.

Week One Day Four: The Chickie Buffer

After starting the ICP- OES, I created 5 mL samples including 50 uL of the samples provided that consist of DNA with 1 mM Cobalt2+ and varied amounts of Sodium+ ions dissolved in a solution of KCl and Tris. The samples had sodium amounts from 0 to 80 mM. The samples were run with the calibration of cobalt, sodium and phosphorus that were created previously. I had the chance to modify the method for this run from the method used yesterday. The ICP was then put on standby for Monday and I have several graphs to make on Monday based on the data from today's run. Hopefully I can put a few of them in on Monday once they are completed.

P.S.

I almost forgot to mention a revelation I had while Prof. Andresen was explaining the physics (mostly thermo) behind what we are attempting to do. For over ten years, studies have been undertaken to determine why, under certain conditions that I don't remember exactly, DNA will attract another DNA even though they are both negatively charged. The Poisson- Boltzmann equation fits what is observed except for the attraction part. In lab, we are trying to evaluate what is causing this attraction. This leads to my revelation, Prof. Andresen explained this to me today and all I thought about was a scene in Chasing Liberty (a movie about the president's daughter on an adventure in Europe) and the part where the president's daughter was a "Chickie buffer (that) negates the potential for man-touching-man discomfort" so that the two guys in the movie could hug with her between them. So this summer, I am trying to find the chickie buffer!

Week 1 Day 4

Today I delved into some LabVIEW code that we will be using for our magnetic tweezers setup. We identified the sub VIs pertaining to motor control and basically figured out how they work. We also identified possibilities for future motor control.

Summer 2014: Magnets, DNA, and Fun!

Abby and Steve hard at work in our newly created student office!

The summer has begun with two great new students (and the same old professor). We have a couple of great projects going on this summer from building a new instrument from the ground up to continuing our quest to understand the physics behind DNA packing! (In fact they have gotten some posts in ahead of me below.)

Week 1 Days 1/2/3

Day 1

I began by familiarizing myself with the programming language LabVIEW. I had done a little bit of programming before this, mostly in MATLAB and java. LabVIEW is very different than these other languages, in that it is a graphical (visual) programming language. Instead of the code being written, it looks like a circuit diagram. LabVIEW has several advantages over traditional programming languages is that there is no real compiler. Errors also tend to be easy to find as the interface won't even let you run the code if there is an error present, and shows you the error. I made several simple programs from a tutorial that introduced LabVIEW concepts. Such programs include one that determines whether or not to hire someone based on their grade, a conversion of a numbered grade to a letter grade, and a decision maker based on a machine's running temperature.

Fig 1: This is an example of the decision maker program. The top shows the front panel of LabVIEW, which displays the output of the code. The bottom is the block diagram, the code itself. For an input temperature and current machine speed, the code determines if the machine is running too hot. If it is not running too hot, it allows the machine to speed up.

Day 2

I continued my familiarization with LabVIEW by writing several programs that created a sine signal and then applied noise to it. One program filtered the noise out and produced a filtered sine signal and displayed both the unfiltered and filtered signal for the user. Another allowed the user to create an rms form of the sine wave and displayed a table of the rms measurements for the user. I also created a program which allowed the user to save data and selected data points of peak to peak measurements of the filtered sine wave to a text file.

Fig 2: This program creates a sine signal and then adds noise to it. The unfiltered sine signal is displayed for the user graphically in the front panel. The signal is then filtered and displayed for the user. The code displays a warning signal to the user for peak to peak measurements of the filtered signal that are above a threshold. The user also has the option to press a button on the front panel that allows them to save certain data points to a text file.

Day 3

I read several papers on the principles and design of a magnetic tweezers (MT) experimental apparatus. I discovered that multiple methods exist for the tracking of the beads used in MT applications. I also familiarized myself with the physics behind MT.

Week One: Day Three

I started up the ICP with help from Prof. Andresen, including putting new tubing on the Peristaltic pump. I ran a calibration set for 50mL solutions comprising of 0-500ppb of Na, Co, and P for a total of six calibrations. These were also used as the samples. In the afternoon, I read through the software manual and then Prof. Andresen and I reprocessed the samples from the morning and reviewed the data.

Week 9

Last week and probably final post for the summer.  Finished that last trial and ran the samples 4 times, averaging.  Then ran the buffer and spin 8 again another 4 times and averaged them in.  Took standard deviation of the data points for my error.  Unfortunately the phosphorus count was very low, and the trend was not defined in the sodium.  But error bars were a little smaller and we were on the right track.

Next, I only had enough nucleosomes for 3 more samples.  So I made the second, third, and fifth sample a last time, spun in the cold room, ran the NCP, buffer, spin 8 and spin 7 many times and averaged them together.  This time the phosphorus count came out well, the trend was defined, and the error bars much MUCH smaller.  Certainly a good place to end for the summer.  Hopefully after Andresen makes more nucleosomes, I can run again in the fall and perfect the experiment, getting reproducible data again and again.  For now I can work on a poster for Celebration next spring, and in the future (maybe?) help get the data published?

Week 8 day 3

Today, I finished analyzing my data from the last two experiments I ran. My second experiment came out very weird, but the data from my first appeared good except for the sodium. Now I am going to run the experiment again to drive down my error bars. Today I made my NCP samples and spun them. I also made a new calibration set because I discovered that my previous data was not covered completely by the previous calibration. Tomorrow I will run the spectrometer and analyze this new data.

Week 8 Day 1-2

In the last couple days I have gone through the steps of another trial.  Spun friday, made samples and ran monday/ tuesday.  This time per Andresen request I ran the samples out, 4 times.  I will average the data and take the standard deviation from those 4 trials.  The I will go through the same steps of analysis to come up with my graphs.  Looks good in the preliminary raw data.  Will see how I feel later on tomorrow.

On another note I ran the chlorine and bromine calibrations I made.  Lets just say the spectrometer didn't detect any difference in the  0 ppm Br and the 1000 ppm Br samples.  The chlorine detected something, but the correlation is really bad.  For both of these elements when you go to look at the spectra in the offline Winlab mode, there really is no peak to see.  Perhaps the spectrometer isn't looking at the right wavelength?  More thought will be put into this.

Lab Instructions

Finished the lab instructions for my experiment with the help of Brian.

Making the Calibration Set:

1.     Know how much of each element you need to cover in the calibration set.  For my purposes we did between 0-0.5 ppm Mg, 0-1.2 ppm Na and P, and 1 ppm Co for 10 mL of water.  Note:  See document 6-26-12 Calib Specs.xlsx .  If you do something different from this, use this formula to calculate how much you will need for your calibrations:

(Required ppm*Required Volume)/(Stock’s ppm)

For example:

I have 50 ppm stock of Mg and want 0.5 ppm Mg in 10 mL of water. 

Calculate:

(0.5 ppm*10,000 micro liters)/(50 ppm)=100 micro liters of Mg soln. needed in my calibration

2.     Please use the 50 ppm stock, if there isn’t enough make more.  Believe me, you do not want to be struggling with 1 and 2 micro liters, working with smaller amounts increases your percent error. 

3.     Use my document, already named above, as a template for your calibration specifications.  Just save as!  I have the spreadsheet set up so it will calculate your final, actual concentrations of each element in your calibrations, which you will need to put into the Method in Winlab for analysis. 

4.     Now just follow the steps in the spreadsheet.  Weigh tubes before and after you add solution and check the percent error before you move on, sometimes you’ll have to re-due one!

5.     Most importantly, run the calibration set in the spectrometer before you use it for analysis.  For 10 mL of calibration, you can run the machine about 4 times.  Look at the correlation in the calibration set, if everything is 0.99-0.999 then you are good to go!  A great calibration set makes a world of difference in the analysis.

Making the Sample Set:

1.     If you have made the calibrations, this is pretty self-explanatory.  Use document 6-27 Sample Specs.xlsx.  This is the template for the same samples I made.  It tells you have much NaCl, MgCl2, Tris, and water to add for 5 ml samples.  The specs on these samples are:

0, 0.5, 1, 2, 3 mM MgCl2

10 mM NaCl in each

1 ppm Tris

2.     Again, this spread sheet is set up to give you your final, actual amounts of element in each sample.  Use this as a point of comparison in the analysis, after you get your data, just to make sure everything came out as it was supposed to.

3.     Now move onto the cold room for spinning!

Spinning Samples in Centrifuge 5418:

Note: Spin samples in the cold room in the science center.

1. Take centrifugal filter tubes and put filters inside of them.

2. Pipette 200 μL of NCP into top of tube.

3. Open centrifuge. Unlock and remove lid by twisting counterclockwise.

4. Put your tubes inside holes.

5. Balance tubes in the centrifuge. For example, if you place another tube in location 1, there also needs to be another tube in location 10. There always needs to be an even numbers of tubes. If there is an odd number, fill another tube with the same amount of water.

6. When there are more than two tubes, the tubes need to be exactly opposite from each other. For example, use locations 1, 2, 10 and 11 instead of locations 1, 6, 10 and 15.

7. Put lock back on. Close lid.

8. Set timer to 10 minutes and RCF to 14000.

9. Hit start. Make sure the centrifuge gets up to speed. Hit stop if the centrifuge starts vibrating and check locations of samples to make sure they are evenly spaced.

10. Wait until spinning ends and lid pops up.

11. Remove tubes. Pipette out liquid from bottom of tubes. Be sure not to contaminate filters.

12. Put filters back in tubes.

13. Pipette 400 μL of sample into top of tube.

14. Spin again and repeat until 8 spins have been completed. After the 7^th and 8^th spin, collect liquid from bottom of tubes in separate tubes. Weigh these tubes before and after pipetting into them.

15. After the last spin, it is necessary to collect whatever is still in the filters. Weigh new tubes. Flip the filters into these new tubes. Put these tubes back into centrifuge. The caps will not fit into the tubes. Place them toward the center of the centrifuge.

16. Spin again for 2 minutes at 2000 RCF.

17. Keep the remaining solution.

The Analysis:

1.     Make the samples to be run in the spectrometer.  There should be four sets of 5 tubes each.  The leftover NCP, Spin 7, Spin 8, and Buffer.  Make sure to record the masses of how much sample and water you add to each tube.  As a guideline, dilute about 50 micro-liters to 5 mL of water. 

2.     Run sample in the spectrometer.  See the instructions next to the desktop computer if you don’t know how to use the instrument already.

3.     Export the data set.  Use template called “Lauren’s”.

4.     Copy and paste your data into the “Raw Data” tab of the NCP Trial Template.xlsx *note delete the repeated concentration column, you won’t need that column twice

5.     Next copy and paste the raw data into their respected tabs, sorted by analyte.  This takes a little while to do the first time, but gets easier each time you do it.

6.     In the “Dilutions” tab enter the mass of the sample and water in each tube for the respected sets.  Then copy and paste the concentrations column of each set into the Dilutions sheet.  Everything will be calculated for you.

7.     Copy->Paste Special the “Average P” values from column J to column K in the “Ion Count” tab.  Column labeled “Average Org. Conc. (mM).

8.     Copy-> Paste Special column I for each set into their respected columns in the “Ion Count” tab.  *Note you don’t need the concentrations from the phosphorus copied, just the sodium and magnesium

9.     Onto the graphs.  Graph the original buffer concentration of the magnesium vs the excess buffer ions per Nucleosome of the sodium and magnesium.  For this part, just pick one of the analytes to graph and remember which one you used to calculate the error bars.

10.  Lastly error bars.  Not going to lie to you here, I can’t give you much guidance.  If you figure out what is on the spreadsheet, kudos.  Otherwise just start from the beginning and go through all the calculations with the standard deviations.  Remember to add in quadrature when you multiply and divide!  For this step I always have to go back and write out what I did, since I can’t seem to come up with a good system of just plugging things in. 

11.  Check out your graph.  How well did your trial go?

Week 7 Day 4

Today, I decided to try to redo my experiment but with different concentrations of MgCl. After making the samples, the concetrations are 0, 1, 10, 12, and 50 mM. I ran out of nucleosomes while making the last sample so sample 3 has only 15 microliters of NCP rather than 41. After making the samples, I spun them and pipetted out the top and bottom into separate tubes. I am ready to run the samples again when we have gas.

Week 7 Day 4

As with all science, you can't just do an experiment once to prove a hypothesis.  So today I made another set of samples just like the last set, to repeat the experiment and see if I can get the same results. Of course I don't anticipate having the same problems with the scale again, so I think this next run will go smoother and I can get results quicker!

Week 7 Day 3

I analyzed all of my data from my experiment and I calculated the difference in Na, Mg, and P from the top of my samples vs. the bottom of my samples. The Mg and P have small differences in the samples with no MgCl(sample 1) and 50 mM MgCl(sample 4). They have much larger differences with 5(sample 2) and 10 mM MgCl(sample 3). The Na has an odd trend.

Here are the graphs of Sodium 589.592, Mg 279.553, and P 178.221. When finding the differences, I took the absolute value. The error bars are too small to be seen. The y axis has units of mM.

Week 7 Days 1/2/3

So far been a pretty slow week, but with good results :)  Monday I waited for the gas guys to come, then purged the instrument all afternoon.  In the evening I ran the samples from last week.

Tuesday I reviewed the data.  Unfortunately back when we made the samples the scale freaked out and started spitting out weird numbers, so I when I put those masses back into the analysis I got back all negative ion counts.  Obviously something went wrong there.  Fortunately the scale was fine when we made the buffer samples, and we don't really care about the Spin 7/8 samples for the graph.  Plus I had enough nucleosomes left to make up another set of NCP samples to run.  So thats what I did.  I made the samples and we had enough argon left to run the spectrometer again.

Today I came in and reviewed the corrected data, and it looks beautiful!  Tiny error bars and the correct trend.  To bad Andresen isn't here to (celebrate? can we do that yet?) and look over the data.  Here are the graphs though, what do you think?

Week 7 Day 1

Today I started on a new project. I started by making samples which will be finished tomorrow, then spun and ran in the spectrometer. I am going to be measuring the difference in NCP concentrations at the top of our solutions and the bottom of our solutions. The samples I made today consisted of 1 mM Trs, 10 mM NaCl, 0,0.5, 1, and 5 mM MgCl and 1 mg/ml of NCPs.

Week 6 Day 3 and 4

Wednesday we made up the samples and were going to run them, but we are out of gas.  Will have to wait to run till Monday or Tuesday.  Instead we made presentations to show andresen and started to write up Lab Instructions for my experiment.

Thursday we presented the presentations, got a little feedback, and Brian moved on to a new project.  I made up some calibration sets with Chloride and Bromine to determine whether the spectrometer can measure those elements.  Once again we do not have gas to run the spectrometer, so will have to wait to look at those sets until next week.

Week 6 Day 2

Today Brian and I continued with the next step of our experiment.  We took turns every ten minutes going into the cold room in the Science Center to do the spinning.  The experiment seems to have gone well.  We will know more tomorrow when we send the samples through the spectrometer.  We were also visited by Dan with questions as to why we were so bundled up on a hot summer day.

Week 6 Day 2

We came in this afternoon and went to the science center to spin our samples in the cold room. It was cold. We will run the samples in the Spectrometer tomorrow.

Einstein Has Joined the Lab

Einstein has come to live in our lab and is a nice distraction with all the sets of clothes he came with.

Week 6 Day 1

Back from break.  Just before we left, we found out that I added phosphorus to the samples and should not have because the nucleosomes already had them.  So this is what messed up our last run.  So today Brian and I remade the samples without the phosphorus.  After we spin them in the cold room tomorrow we will add the 1 ppm Co standard.  Then into the spectrometer on Wednesday!  Hopefully this run comes out perfect.

Here is a fun picture of our samples

Week 6 Day 1

After a week off, Lauren and I came in this afternoon to resume our experiments. We are planning on redoing our experiment from 2 weeks ago. Today we made samples with MgCl, NaCl and Trs. We did not include P. Adding P messed up our results last time. We are planning to spin the samples tomorrow and will run the samples on Wednesday in the spectrometer. To prepare for this, we also labeled and weighed tubes for tomorrow. We will keep the sample after Spin 7, Spin 8, and after we flip our filters.