Author: Prof. Andresen

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Week One Day Four: The Chickie Buffer

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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!

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Week 1 Day 4

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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.

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Week 1 Days 1/2/3

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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.
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Week One: Day Three

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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.

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Week 9

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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?

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Week 8 day 3

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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.

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Week 8 Day 1-2

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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.

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Lab Instructions

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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 7th and 8th 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?

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Week 7 Day 4

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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.

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Week 7 Day 4

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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!