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.