Agarose Gel Electrophoresis!

The past two days, we have prepared and ran 2 gels to determine the best digestion for the chromatin. I added various amounts (from  5 to 80 Worthington units/mg chromatin) of micrococcal nuclease to small samples of the chromatin solution. We made sure that the concentration of DNA in the solutions was less than 100ng/ml so that the gel would run without smudging. We mistakenly let the first gel run overnight at a low voltage but it over ran. Today we ran it through lunch until the visible blue line was at about 2.5 cm. We determined from the imaged gel that 15 Worthington units/mg chromatin of the micrococcal nuclease is sufficient. I then digested all of our chromatin and centrifuged it down. Tomorrow we will be doing a column and another gel to see if our nucleosomes are adequate to use for experiments.

Gel electrophoresis set up including the black death stuffed toy that watched to make sure our gel ran smoothly.

  

The End is Nigh

This is the last week of my summer research...

A bit dramatic but I'm doing equilibrium dialysis right now so I feel Frodo's pain ;)

 Last week I did the nanoparticle titration with sodium chloride and found that partial aggregation occurs at 100 mM and complete aggregation occurs at 200 mM. Then I did a dialysis of pure PSS in sodium chloride concentrations up to 50 mM and ran DLS and ICP on the resultant samples. The DLS showed some weak trends that may indicate a change in the PSS conformation dependent on salt concentration. The ICP results showed no difference in the amount of Na/S at different salt concentrations.

This week I am running an equilibrium dialysis on a new batch of PSS coated nanoparticles against a few sodium concentrations up to 50 mM. I got a new dialysis system that holds 4 mL at a time so I can run the same amount of sample in fewer tubes. Each concentration has 2 tubes, one will undergo both the salt and water and the other just the salt dialysis. This way we can run DLS on the samples that still have high salt concentration while running ICP on the water dialyzed samples without having to worry about background sodium.

This week, I will only have time to do the dialysis and the complete workup for it. I'm going abroad in the fall, to Lancaster University England, but I am looking forward to continuing this research in the spring!

Preparing Nucleosomes!!

This week Professor Andresen and I have been preparing mononucleosomes from chicken erythrocytes. Hopefully by Tuesday we will have clean, pure mononucleosomes that we can then use to investigate their electrostatics. So far we have taken the nuclei out of the blood, cleaned it, digested it with micrococcal nuclease and separated the DNA/nucleosomes from the nuclei.

July 10th - Computer Simulations

Recently, I have been attempting to model DNA-ion interactions through a computer program called Delphi. After imputing atomic coordinates, atomic radii, and atomic charges files, Delphi solves the linear and non-linear Poisson-Boltzmann equations and can output the charge neutralized by the ions (can be compared to the charge of the DNA) as well as three-dimensional concentration maps.

Even more recently, I used Matlab to generate a hexagonal array of 19, 60 base pair long, DNA strands (see below). In our experiments the DNA was condensed into a hexagonal array, so these generated atomic coordinates produce a more accurate simulation of the experimental DNA-ion interactions.

Now, I will be working on adjusting parameters in Delphi (grid size, ion valence, ion concentration, boundary conditions, etc.) to match what is expected of the system (almost if not complete charge neutralization of the DNA) to the actual simulation results.

Reproducible Results!

So many dilutions! Endless labelling...

Last week, I repeated the equilibrium dialysis two separate times and prepared calibrations and dilutions for ICP.

I wasn't able to run ICP until Monday since we ran out of Argon gas. I ran the ICP on Monday and Tuesday (only 2 runs because my samples were limited). After analyzing the data, we found that both dialysis runs showed 1 Na/S and nearly 1 K/S! I found a calculation error in the data analysis from last week which, when corrected, indicated that both Na and K samples had 1 ion for every 4 S. That dialysis was a "quick and dirty" experiment so we are more confident in the most recent data which has proven to be reproducible.

One thing that looks interesting is that the hydrodynamic radius of the nanoparticles goes down after salt dialysis and back up after water dialysis. After the water dialysis is can be up to 30 nm bigger than after the salt dialysis! Since the amount of PSS/NP is remaining constant, this data indicates that the conformation of the PSS is changing in response to the salt concentration.

Aggregated Nanoparticles

Going forward, we are going to run some tests with varying salt concentration. Today I did a titration of NaCl into the PSS coated NPs and took samples at a range of salt concentrations. I found that the nanoparticles aggregated (due to instability) partially at 100mM salt and completely at above 200 mM salt. This will give us an idea of what range we can dialyze the nanoparticles at.

Back the the Lab!

After spending about six months in Australia not doing any physics, I'm back and ready to start a new research project that will hopefully culminate in my capstone project! Instead of working with DNA, I will be conducting experiments using mononucleosomes that Professor Andresen and I will be trying to prepare over the next few days. The protocol looks very scary and full of biochemistry to prepare, clean and inspect the chicken blood until its just nucleosomes but hopefully we will succeed in preparing them ourselves so that it will open many research opportunities in the lab. Wish us luck!

Error Bars

The ICP data from the sodium/potassium trial had extensive error bars due to the large range of sodium and potassium concentrations tested. It was impossible to measure a difference of a few ppm over top of a background concentration of 300 ppm. To resolve this I dialyzed some of the nanoparticles from the previous dialysis with water to clear out the background salt and leave only the gold nanoparticles with anything bound to them. The ICP data for those samples had much smaller error and very interesting results. It showed that there was 1 Na for every 4 sulfurs and 1 K for every 7 sulfurs. Going forward I am going to repeat this process with the water dialysis a few more times get more data and confirm that the data is repeatable.

Before Water Dialysis

After Water Dialysis

Sodium v Potassium: Round 1

On Monday, I started making a brand new batch of PSS coated NPs. I still have a good portion of the last batch but I wanted to make sure that the data we were seeing was reproducible. On Tuesday, I characterized these particles and ran equilibrium dialysis on them with NaCl and KCl in parallel. This way, I hope to compare the absorption of Na and K ions. Today, I characterized the resultant nanoparticle samples and ran all the samples in the ICP 4 times with 2 different calibrations. If the data is reproducible I hope to see 4 Na per S like last week's run. The charge on all of the dialyzed nanoparticles since I modified the protocol to dilute with the dialysis buffer has been negative, which matches our original expectations.

June 24th - Data Analysis

For the past week or so I have been working on analyzing the data I gathered from ICP-AES. So far I have found two interesting trends: as the PEG percentage increases (so, DNA-DNA spacing decreases), the charge neutralized by hexamminecobalt(III) increases and the charge neutralized by magnesium decreases, and as the magnesium concentration increases, the charge neutralized by magnesium increases and the charge neutralized by hexamminecobalt(III) decreases. These two trends are not surprising, and correlate well with the expectations of the DNA system. What is even more interesting though, is determining the concentration of magnesium needed at each PEG percentage so that hexamminecobalt(III) and magnesium equally charge neutralize the DNA (shown in the graph below).

ICP

Yesterday, I ran another equilibrium dialysis on the nanoparticles using NaCl. I modified my protocol to dilute the final nanoparticle product with NaCl instead of pure water. Today, I characterized these nanoparticles. They were negatively charged this time which is what we would have expected but we still don't know why the other two samples are positively charged. This afternoon I am running 4-5 ICP runs on the samples, which I have diluted. This will hopefully give me good statistical data to analyze.

Just watching the autosampler do all my work for me...

Too much sodium!

Yesterday, I ran another equilibrium dialysis with the same parameters but I doubled the number of spins to 20.

Today, I characterized the resultant nanoparticles, prepared new calibration samples, and ran all of the dialysis samples in the ICP. I diluted the nanoparticle sample to attempt to lower the concentration range the machine was reading. There were a few interesting results. Firstly, both this sample and the last sample are reading positive charges. The polymer coat is negatively charged so the only possible explanation is that the excess sodium (3:1 Na:S in the first sample and 6:1 Na:S in the second) is causing the surface to become positive. The very presence of an excess of sodium is counterintuitive, one would assume that the polymer would only absorb enough sodium to neutralize its charge. Going forward I will be doing more ICP and dialysis to get more data. 

The Files are Breeding Like Tribbles!

On Thursday, I ran ICP on the uncoated nanoparticles and the coated pre-dialysis nanoparticles. Since  then, I have spent most of time trying to analyze the ICP data and reading literature on polyelectrolyte deposition. As a result, I have 7 excel documents open on my computer and even more pdfs!

Next week, I plan to run dialysis on the same nanoparticle sample with KCl as the salt buffer. I will then run ICP on those samples so that I can compare them to the nanoparticles that underwent dialysis with NaCl.

June 10th - Compiling Data

These past few days I have been working on compiling the ICP-AES data from multiple runs. Below is one set of the graphs that compares the results from each run. Because I am investigating the competitive binding between hexamminecobalt(III) (which has a charge of +3) and magnesium (which has a charge of +2) to DNA at varying levels of osmotic stress, the graphs below demonstrate how much of the DNA charge is neutralized by each respective species at differing PEG values. It is interesting to note all runs seem to have the same trend, and hopefully with even more data the numerical values for the trend will become more solidified. Tomorrow, I plan to run another set of samples through the ICP-AES and compare its results as well.

Dialysis

Yesterday, I finished making and characterizing a large batch of PSS coated NPs. I then tried to run equilibrium dialysis. I modified my parameters, decreasing both time and rotation speed, to hopefully correct the loss of concentration. I successfully recovered about 1.7 nM which is a big improvement over last week.

Today, I characterized the new nanoparticle sample, prepared new calibration standards, and ran ICP on the equilibrium dialysis samples.

Tomorrow, I plan to do analysis on the ICP data I have acquired and possibly run ICP on the non-dialysized PSS nanoparticles for comparison.

Sulfur!

On Friday, I ran equilibrium dialysis on my PSS coated nanoparticles using a centrifugation system. Unfortunately, a significant amount of the nanoparticles stuck to the mesh in the filter causing a significant reduction in concentration. This was a problem since we needed a relatively high concentration in order for the ICP to be able to detect sulfur, an element found in the PSS coat.

Today, we came up with two solutions. First, we purchased a different type of dialysis device that floats in water and allows equilibrium to be reached over time.

The new dialysis device will take over a week to arrive so, in the meantime, I concentrated the nanoparticle solution from Friday's dialysis and ran it in the ICP. In the concentrating process, we lost a good deal of volume so we had to use a couple tricks to get the machine to use up less sample. From my calculations, we were not very optimistic that the machine would be able to detect the sulfur. However, much too our surprise, the sample had more sulfur than even our highest estimate. Tomorrow we will look at the official ICP report and it should be able to tell us something about the effectiveness of the dialysis and the ion atmosphere around the nanoparticles. Also, I will try to work out some better parameters for our existing dialysis system.

June 5th - Lab Work

After successful preparation of calibration standards yesterday, I made 86 samples (below) to run through the ICP-AES next week. Next week I will also be preparing more calibration standards and samples.

Polystyrene Sulfonate

Today, we ran our calibration samples in the ICP-AES to get an idea of how precisely we made our samples and to learn how to use the machine. After we finished running the samples and analyzing the results, I visited Professor Thompson's lab in the Chemistry department where he outlined the process we will follow for the next couple weeks. Today, I diluted a solution of spherical gold nanoparticles he prepared, and treated them with polystyrene sulfonate and sodium chloride. This mixture needs to sit over night so the polystyrene sulfonate has time to absorb onto the nanoparticles surface. Tomorrow, I will centrifuge down the treated nanoparticles and begin characterizing both the treated and untreated samples.

June 3rd - Summer Research Begins!

Looking forward to an interesting and exciting summer! These past few days I have been organizing and gathering background information by reading scientific papers so I can begin research soon. Today we ran standard samples through the ICP-AES to learn how the machine works as well as determine the precision of our standards. Hopefully later this week I will be able to run some samples through the ICP-AES and begin to look at ion binding competition in DNA-arrays!

The epic quest begins...

Today was the first day of summer research! Ambitious plans were laid, literature was read, and calculations were made. My project will focus on the interactions between gold nanoparticles and DNA. My goal for the next couple weeks will be to synthesize polyelectrolyte coated gold nanospheres and characterize their features. Then I will run equilibrium dialysis and atomic emission spectroscopy (ICP-AES) on these nanospheres with different concentrations of ions. I'm looking forward to an exciting summer!