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