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!