Andresen Lab Blog

This morning I finished my calibration samples. I also noticed a mistake in my buffers so I had to remake them too. After I did this we had our group meeting in the physics lounge. Dr. Good had some Good pizza. After lunch I put nucleosomes in new samples and spun them in the centrifuge with my buffers and then water. I did four spins with the buffers and 5 spins with the water. The water was supposed to only be 4 spins but I accidentally spun them a fifth time. When I retrieved my solutions after spinning, I weighed to find the amount of nucleosomes. My first sample had .0232g and my second sample had .0044g. This seems like a very large separation. I also added cobalt as an internal standard to the solution and filled up to 6 ml with water.

Today we decided to remake the sodium samples and run them again, since it seemed that there was such high correlation in my trial runs that did not match my expected values.  So thats what I did.  I re-made the samples this time by first making a concentrated solutions and then diluting it down to the concentrations I wanted.  At thank god it mostly worked.  My percent errors went down significantly.  A good way to end a Friday!  Of course, I’ll see if I can play around with this sodium data a little bit more to see if there is a trick with getting the error lower.  Till funday monday!

This morning I ran the spectrometer. When I got the results they were all over the place. However I forgot to account for the fact that there was no Cobalt as an internal standard for my nucleosome solutions. After I did this, the data did not look good so we are going to run the experiment again. This time I will keep the liquid that comes out during spinning. We will look at this to see if the nucleosomes come out of the solution. In the afternoon I remade the buffer solutions and started remaking the calibration samples. Tomorrow I will finish the samples and spin them.

So today I ran three of those tests I was working on with the sodium.  Didn’t even bother running the 4th because we were getting consistent data….that didn’t match our actual concentration.  We tried analyzing it the normal way, and by taking away the calibration.  That didn’t help.  So possibly it is because I calculated the concentrations in my sample wrong, since the spectrometer DID give us consistent calculated concentrations across trials.  So now I’m going to do a weighted average of each wavelength and sample to get one graph with error bars.  Then I am going to try remaking my samples to see if that was an issue.  I’m also thinking in spare time to do a little internet research…there must be some other frustrated scientist out there working with this piece of equipment who had the same issue and found a solution…..

sorry about not posting yesterday.  Basically what I did for the past two days was make up one large set of samples and 4 sets of calibrations.  Took a long time.  What we want to know is exactly how well our spectrometer measures sodium, since so far it doesn’t seem very good.  I made up large samples with NaCl concentrations and cobalt and then calibrations with Na and Co.  I then ran the first of the four sets we will run.  Haven’t looked at the data yet, but seeing as Brian and I are sharing the spectrometer tomorrow, I’ll have time to review it then.

A note about my awesome color coding skills.  All the stands that the sample and calibration tubes are set in match the color strip in its corresponding excel document 😀 how’s that for organization?

Today I spun my samples in the centrifuge for the first time. I spun the nucleosomes 4 times with the samples I made a few days ago. Then I spun the nuclesomes with water 12 times. This took me until past lunchtime. After a spun the samples I put them into tubes and put in about 6 ml of water. Tomorrow I will run the samples in the spectrometer. I would’ve done that today but Lauren was using it.

This morning I made the axial align solution for the spectrometer. In the afternoon I ran the spectrometer with the samples I made yesterday.

The data came out well except for the sodium. The correlation coefficient was around 0.916 after initial reprocessing. I tried to fix the data by deleting sample 2. This sample had 0.049 ppm of Na. The spectrometer read it as having negative sodium. However the data did not look any better after deleting the point. The correlation coefficient was 0.90. Na 589.592 gave the worst data. It gave a negative value for Na when there was 0.33 ppm in the sample.

The correlation coefficients of the Mg and P were better. The Mg was about 0.98 and the P was greater than 0.990.

This morning I made the buffer solutions. I made 4 of them. In the afternoon I had to make more calibration samples because I made a mistake in my previous ones. I also added Na to these samples.

Today I continued to analyze data.  Fixed problems andresen found within my excel spreadsheets, and then spent the afternoon on error propagation.  Long tedious work to say the least, and it is almost completely impossible to come up with a way of organizing the error propagation steps in an excel spreadsheet, but i’ll take another stab at that tomorrow when i’m fresh.

This morning I finished making the excel spreadsheets from yesterday pretty.  Also doubled the NCP dilutions for the mM concentrations because originally AP and KA diluted it to 200 ml instead of 100 ml accidentally.  Next I found the excess ions per nucleosome.  For this calculation the average P was the average of the two P data sets from the NCP data.  At the end of the morning we had a department meeting just to share with everyone what we are doing this summer.  In the afternoon I went over the data with Andresen.  It looks like it isn’t good.  As the Na decreases, the Mg should increase.  But according to the data the Na and Mg increase together.  So I moved on to the second set of data.  File labeled Nucleosome Trial 4-AP.  I followed the same steps as last time, except this time I already had the excel spread sheets, so I just copy pasted stuff in and then made it look pretty.  Will talk about the results with Andresen on monday.