Last Week

So last week Monday morning came around and Professor Thompson had been too busy to prepare gold nano-particles for me. This was fine, I was ready to tackle the task on my own. The procedure involves bringing 100mL of a solution up to a boil and then pouring 3mL of another solution into it. The solution then proceeds to change colors several times, its actually pretty neat to watch. I ended up making a pretty concentrated batch of nano-particles. It even ended looking pretty good under the UV-Vis, so I decided to continue on with my project using them. I needed to find a good ratio of NPs and PAH because the two are pretty fragile. If not mixed together properly they can immediately aggregate and make the solution unusable. Also, if they are not in the proper ratio this can also cause it to become aggregated and then unusable. So I tried to make a 3x and a 5x diluted NPs solution with normal concentrations of PAH. Both seemed usable after mixing. I then tried out the new cleaning procedure that Professor Thompson had suggested. I would spin the solutions at very high speeds, 9000 rxg and 7000 rxg in order to get all of the excess PAH out of the solution. This maximized our risk of contamination by stray PAH particles. My problem with this procedure arose when I had to wash the pellets with DNA in them. After I started this centrifugal process, I found that the solutions were creating pellets that were virtually non-existent. This is not good, as it is one of our main goals to get good pellets here. I was thinking that these pellets could not form because I had diluted the initial amount of NPs in the beginning. Professor Thompson suggested it had something to do with my spinning procedure for the DNA washes. I'm now currently trying this new procedure, which includes longer times and a larger rxg, to get better pellets in my solutions.

Continuation of Week 6

Is finally Friday! Well that doesn't matter, what matter is what I have accomplish this week. In terms of work, I have accomplish  a great deal but in terms of progress I could had accomplish more. I say this because we got our results from Monday and we didn't get the results we wanted... again. This is really frustrating but like I said last time, researching is about learning from our mistakes and improving upon them even if it takes multiple failures. Once we saw our results, professor Andresen and I came out with a new method to try out it order to see if we were doing our trial digestion correctly. Instead of using our chromatin, we instead used sonicated DNA with our trial digestion to see if we get different results in our gel. In order to do this we must first shred DNA and we would be left with a solution of liquid DNA.Then I added 50 micro-liters of sonicated DNA into two micro-centrifuge test tubes. Once this was done, I diluted the micrococcal nuclease to 100x dilution and also diluted CaC12 in order to add to our two samples. I added 0.5 micro-liters of micrococcal nuclease to one sample and to the other sample I added 2.5 in order to have two different lengths of DNA. Once this was done, I put both solutions in the heater for 15 minutes at 37 degrees Celsius and when this was done I did the same procedure as the previous trial digestions. In the end had two different samples to test in our gel, but I also made some extra samples such as DNA ladder and just the shredded DNA and the same samples with just added DI water. I added this solutions into our gel,  and when the results were ready, we again got the results which was more frustrated because it still didn't work. The results can be seen in the next picture. Figure #1

Figure #1: Results from Gel

From this picture, we can clearly see that we can't  see any base pairs from our samples, only from our DNA ladder which is located to the left. This is the problem that we have been getting on our past trials and from this picture, we think that all of our samples are staying in the beginning of gel. In other words our micrococcal nuclease is not separating our chromosomes.
After this process was done, professor andresen and I decided to do one more trial with the past solutions but this time, instead of making 3% agarose gel, we will be making 1% gel. What this would do, it would make us see the base pairs stuck at the top of gel from the previous samples. For this new trial, I will use two samples with out the proteinase K trial digestion and two other samples with the full trial digestion. Unfortunately when I was moving the agarose gel to the special the machine that was gonna give me my results, the gel felt apart and what this meant was that I needed to redo my work all over again. which I eventually did.
The next day I ran the gel and while I was waiting for the gel to be done I prepared another trial digestion that I will do next week. This new trial digestion consisted of time intervals of 5, 10, 20, 40, and 60 minutes and 1.6 micro-liters of 100x diluted micrococcal nuclease.
Once the gel was done, I look at the results and they were a little better than previous results but it still didn't show the base pairs that we want it to see. We are hoping that next week with this new trial digestion, we will be able to see the results we want to see. Its a struggle keep seeing how our trials always fail but even though its slowly, we are making progress and we will be able to find what is causing our results.

End week six

The last run of samples was supposed to be our most promising samples yet. It had looked like we were going to get not only quality data but in a large quantity as well. Unfortunately, something had gone awry. When we start with out citrate nano-particles they should be negative in charge, then we wrap them in PAH and they switch to positive charge, and then finally we wrap them in DNA and they switch charges a final time, back to negative. My first two Zeta tests were conclusive and yielded the expected results, however the final one simply doubled the number and did not switch the charge at all. Professor Andresen phoned a friend and we then scheduled a conference with Professor Thompson. Before we could have this conference however, I first had to prepare a power point presentation on my most recent bit of research. Dr. Thompson broke down several of the data pieces I am so accustomed to collecting and helped me better understand them. The UV-Vis is a lot more helpful then I know it to be, but I really need to learn how to overlay graphs on it, I am determined to learn this early next week when I have to use the machine again. The conference left me feeling very hopeful about the project. On Monday Professor Thompson is going to prepare more nanoparticles for me to use in my next batch of samples. He also suggested a much more appropriate way of preparing the PAH wrap and how to more properly purify them. I am hopeful that this will greatly improve the project. At the end of this week, I am wrapping it up by shearing DNA and looking forward to finally getting proper data next week.

Week six

So at my last post I had just found an appropriate mix of salts and nano-particles. This allowed me to then mix in my DNA and start performing equilibrium dialysis. However, I immediately ran into another big problem. What was I to re hydrate my solutions with? In the past, at this step I was not using citrate nanoparticles, but I was re hydrating with TEM buffer. I was uncertain if this would be efficient with these new particles. I decided to head to the science center and discus this problem with Rich, a fellow user of similar nano-particles. We threw a few ideas back in forth, but nothing seemed like an exceptionally good idea to re hydrate with. So I had to make a sacrifice, I gave up any hope of getting to use the ICP-OES this run in order to figure out what to re hydrate my samples with. I split my total solution of nano particles into three parts, one would be re hydrated  with mili-Q, another with TEM buffer, and a third with a solution that was 10^-2 M NaCl. Unfortunately I was only re hydrating with 4mL each when I typically do double that. Regardless, the results appeared to be conclusive, showing that the salt buffer worked the best.

So then I had to restart once again. This time, I took all of my gold nano particles and coated them with PAH. I then spun them in the centrifuge for an increasingly long period of time to siphon out the supernatant and continue spinning that. Finally, I recombined the pellets and diluted it back to 7mL with more nano particles. So I'm left with 1 super concentrated gold nano particle sample and 7 other slightly less concentrated samples. They were then all mixed with DNA and left to sit over night. Now I am currently trying to perform equilibrium dialysis, and hopefully after doing this I'll finally be able to use the ICP-OES.

End of fifth week already?

Time surely flies by when you're working full on. This week, I started working on another batch of ITC experiments after we figured out a number of flaws in our experimental protocols. Therefore, we decided to redo some of our previous experiments using different experimental parameters and protocols.
We decided that we would keep the relative concentrations of DNA and Cobalt Hexammine unaltered in the different ITC runs. The concentrations of DNA and Cobalt Hexammine used in the experiments were 2.5mM DNA+3mM Cobalt Hexammine, 5mM DNA+6mM Cobalt Hexammine, 10mM DNA+12mM Cobalt Hexammine. In addition, we decided to alter the experimental procedure a tad bit. We decided to form isotonic DNA+NaCl solutions using the dialysis method so that the concentrations of NaCl in all the samples are equal. The dialysis buffer was used for the serial dilution of the 120mM stock Cobalt Hexammine (instead of water as in the previous experiments).

After I prepared the batch of DNA samples (of different concentrations) using the regular method, I filled up dialysis tubes with 10ml of each sample. I left the tubes in a dialysis buffer of 10mM NaCl solution overnight. I used the same dialysis buffer for the serial dilution of the 120mM stock of Cobalt Hexammine in order to synthesize 10ml of 12mM,6mM and 3mM solutions respectively.

After the synthesis of the DNA samples and the Cobalt Hexammine solutions, I ran heat of dilution tests on the Cobalt Hexammine samples. Here are the results:

After running the heat of dilution experiments, I proceeded to start ITC runs with my DNA samples. I started off by using my 10mM DNA sample. Here's the overlay for the raw heat data and the integrated heat data:

That's all the highlights from week 5 folks. Stay tuned for more ITC results next week!

6th week, New trial digestion with some changes.

Wow! it's the sixth week, seems like time never stops and always keeps moving. Well today is a Monday, which are never a good feeling but we gotta hang in there. I started my day by going over the results with Professor Andresen, in order to come out with a solution or a new procedure. Eventually professor Andresen did came out with a new procedure which is the same procedure has before but with slightly different trial digestion. Instead of creating different samples of our solution by adding different amounts of micrococcal nuclease, we instead only made two  50 micro-liters of solution which contain different amounts of micrococcal nuclease. I places this solutions into the heater for 37 degrees Celsius and took out 5 micro-liters of each solution at a specific time. The different times that we assigned were 5, 20, and 40 minutes, with another sample that lasted about 2 hours. This procedure accomplished the task of creating different sizes of DNA  by letting the microccocal nuclease eat up the nucleic acids and taking small amounts of sample at different times and stopping the process by adding EDTA. Once this process was finish, I continued the trial digestion with the same procedure as before by using proteinase K and SDS. In the mean while, I created a new gel in order to put the trials in tomorrow. I also made more TBE buffer to used the next day. All this procedure took approximately the whole day due to the unfortunately broken wrist I got. All is well and I could still work, and that's really important to me because I never give up on something I start.

Final day of Week #5. Finally able to see Results

Its Friday! It is always good to come to work on a Friday because you feel at easy with yourself and work just for the fact that once this day is over, you can rest for two whole days.
Well I started my day out by making more TBE buffer, which consist of 50 ml of TBE (Tris-Borate-EDTA buffer) and 450 ml of DI water. Once I was done making this buffer I made two DNA ladder, DNA ladder is a solution of DNA molecules of different known lengths and is used to run along side our samples in order to estimate the size of our samples. Once this was done, I prepared an extra micro-centrifuge tube with only 1 micro-liter of our chromatic supernatant, 9 micro-liters of 60% sucrose and 10 micro-liters of DI water. I made this extra sample in order to see how it looks on the gel without the trial digestion. For the solutions who already had already been through the trial digestion, I took 1 micro-liter from each and added them to new micro-centrifuge test tubes with the same numbers label in order to not get mix up. I also added 9 micro-liters of 60% sucrose and 10 micro-liters of DI water to the new test tubes. Once this was done I was ready to add them into my gel. The solutions on the gel can be seen in figure #1.

Figure #1 Samples added to Gel

Once I loaded all of my samples into the gel, I set the voltage to 100 volts and waiting until a blue line reached 5 cm. While I was waiting for this process to happen, I started to unpack all of the materials that we ordered in order to have more stock available to used in the future. It was like Christmas morning, unpacking all of the boxes. See figure #2 

Figure # 2 Material needed for present and future usage

Once the unpacking was done, our shelves finally looked like they had material in it and it looks awesome. See figure #3

Figure #3 More beakers to make more Buffer

When all of this was done and my gel was finally done, I took the gel and used a special UV light to the results. Unfortunately once again we didn't get the results we wanted. After I saw my results, I felt disappointed because I had worked so hard to get better results than last time but that didn't change the outcome. Like I said before in previous blogs, being a researcher is about working hard and trying to solve problems even if it means failing multiple times. It is hard to accept the results but my results just show me that I must work harder and try again until I get the results I want. 

Week Five

The results from the last experiment I mentioned in my last blog post were inconclusive. Along with the next few experiments that had occurred after that. Now I'm not saying that it is impossible to get the data that we wanted to get with the materials that I was using, however, Professor Andresen pointed out that if the experiment was this finicky then no one would care about it enough to try to reproduce it. So later that day he found me a brand new batch of nano particles. This time we got Citrate coated gold nano particles and this changed our procedure slightly. The general idea now is to take the negatively charged gold nano particles and wrap positively charged PAH around the gold. Then we'll wrap the DNA around the PAH/Gold complex. I was very excited to get away from the old procedure that I couldn't figure out how to fix. The first problem we ran into with this new experiment was trying to coat the gold with the PAH. When I followed the old procedure something strange happened. 

Starting from the left is a 10^-2M NaCl solution, then a 10^-3M NaCl solution mixed with 10micrograms/mL of PAH, then Batch 4 of the citrate gold nanoparticles, and then on the left is a solution that mixed all of the previous three. It was very concerning that the final solution turned out purple. Something was definitely wrong, so I went to Fontaine to seek help. We tried every combination of her and my particles to see where exactly my purple solution had came from. We could not recreate the purple solution but we did eventually come out with a solution that would allow me to move onto the next step. At this point in time i'm about to mix my new Gold/PAH solution with some sheared DNA so it is looking exciting! 

5th week, final steps on the preparation of Mononucleosomes

When I came back on Wednesday, I took out my solutions from the refrigerator and stop the spinner. This was a struggle but not because it was hard removing the equipment from the refrigerator but for removing it with a wrist paint I felt during this process. This was due to the accident I had with my skate board but as a hard worker I still manage to complete my task and keep working. Once the solution was out of the refrigerator, I put the solution into a special test tube which you are able to see in figure #1 and you are also able to see my wrist support that one of my lovely friend let me borrow.

Figure #1 Special test tube for Centrifuge, this is test tube is used when using a powerful Centrifuge that goes faster than 3900xg

I set the centrifuge to a speed of 5000xg for 5 minutes and after this was done, I removed the supernatant and measured the Concentration of DNA using the UV-vis equipment. From our collected data, professor Andresen and myself found our that we lost about 300 mg of chromatin from our previous UV-vis sample but that is to expected when doing this kind of washing and processes. Once this was done, I dialyses my supernatant against 10mM Tris-HCI and 1mM  CaC12 at 4 degrees Celsius. I was only able to create three dialysis bags for my supernatant due to the fact that my supernatant volume was a lot, the rest of the supernatant will be placed in dialysis bags the next day.
The next day I came back, I removed the dialysis bags from the buffer and created the rest of the dialysis bags for the rest of my supernatant. Once this was done I started the process for the Trial Digestion of my mononucleosomes. I prepared six different microcentrifuge tubes and label them with different numbers, shown in figure #2.

Figure #2 Two rows of different samples and different concentrations of Micrococcal Nuclease

Figure #2 shows two different rows of solutions, one solution was made up of 50 micro-liters of our supernatant and different concentration of Micrococcal nuclease. Once again Micrococcal nuclease is used to digest nucleic acids. We set this solutions to heat up for 55 minutes at a temperature of 37 degrees Celsius and once this is done we removed them from the heater and add 0.5 micro-liters of EDTA to stop this reaction. When we are done with all of this, we again prepare six other micro-centrifuges tubes with the same numbers but with an extra PK on the side to represent Proteinase K. This new tubes contain five micro-liters of our digested chromatic solution, five micro-liters of proteinase K, and 0.5 micro-liters of SDS. We then set this samples into the heater for 50 minutes at 50 degrees Celsius. Once this is done, we can placed our samples in the refrigerator to be used for the next day. During the time I was waiting for my solutions to heat up, I was making new Agarose gel in order to used it the next day and placed my samples in it. 

Today was a really productive day and a really hard one too, this is due to the pain I felt in my wrist after my tragic fail in the skateboard. All is well though, because only one person saw me fall and that doesn't count as a fall. To be honest with this wrist support my hand looks awesome!! but it doesn't mean it feels awesome. On the bright side, I am getting better and I have completed the work for this past days no matter how painful it was and I am proud of that. 

Figure #3 Never give up! even if you feel like your hand is going to fall. 

5th week, Continuation of the preparation of Mononucleosomes

I started this new week by re-suspending  my nuclei in 8ml of ML and spind it down at 3000xg for 5 minutes. I did this process 4 times in total and all of this is done for the preparation of Mononucleosomes. Once this was done four times, I removed the supernatant and took a small sample in order to check for how much DNA concentration I had and added 2M of CaC12 to make 1mm. Once this was done I need to make a calculation of how much Micrococcal nuclease to add to each sample. Micrococcal nuclease is used to eat up the DNA strands until it reaches  the nucleus. Once I found the correct amount of Micrococcal nuclease to add, I pre-equilibrated  the solution to 37 degrees Celsius for short DNA fragments for 30 minutes. Once the timer is up, in order to stop the reaction, I must add 10mm of EDTA, for my specific amount of solution, I added 161.488 micro-liters of EDTA. When all of this process was done, I was once spun the solutions at 1000xg for 5 minutes at 4 degrees Celsius. The final I step I needed to do was to create dialysis bags and let them sit over night, In my case I made 4 different dialysis bags, 3 containing our supernatant and the other containing the nuclei (left over white solution). The next pictures show the tools used to make the dialysis bags.

Dialysis  tubing and Clips.

Dialysis bag 

Dialysis bags, all inside 500mL of EDTA. This must be left over night.

Dialysis tubing and clips are used for the separation of small molecules from macro-molecules.

The next day I came back I took the dialysis bags out from the EDTA and put the 3 liquid samples into test tubes and the white nuclei I also put it in a test tube. The next thing I did was to spin each solution in the centrifuge for 8,000xg for 20 minutes. Once this was done I took out the supernatant and calculated its DNA concentration for each. The next thing I did was to combine both my supernatants together and then calculated how much DNA concentration was in this new product. When this was done, I added 1875 micro-liters of NaCl, 1800 micro-liters of EDTA and .9 grams of CM Sephanex. Once these components were added, I let the solution stir slowly at 4 degrees Celsius for the night. I felt really proud of myself because I was able to due all this solutions by myself. In the previous preparations of these same solutions and procedures , I was learning from the great skills that Professor Andresen had demonstrated and now I am capable of doing such great task. I have learned so much this previous weeks and I am still learning a lot from my mistakes and results. 

When 30-day free trial comes to your rescue

For the past few days, I have been trying to figure out a way to average my data from the NanoAnalyze isotherms. I have tried using different ITC analysis programs to facilitate my work, but to little avail. I started off by downloading a series of ITC based software with some interesting abbreviations- NITPIC and SEDPHAT.

NITPIC can be used to extract the raw ITC data from NanoAnalyze to perform a number of functions including thermogram reconstruction and processing integrated isotherms from the raw thermogram. I ran a test trial using data from my 5mM DNA-Cobalt Hexammine data. The program succeeded in producing a beautiful isotherm for me. I then converted this isotherm into a format that is readable by SEDPHAT. SEDPHAT processed this data and subsequently produced a global fit. However, I still couldn't figure out how to integrate this data with isotherms from my other trials, in order to generate an average fit for my data points. I played around with the program for hours to figure something out. But this is one of those stories without a happy ending.

NITPIC 

SEDPHAT

However, when there's a will, there's a way. I discovered another way to generate a mean isothermal plot. This process was lengthy and relatively complex.

I found a graph plotting software online that has been programmed to produce sigmoid fit for a data set. This software is called OriginPro 2017. I downloaded a 30-day free trial version of the program from a website. I had to average the area data from the "5mM DNA w Cobalt Hexammine" ITC runs using an Excel worksheet and I exported the mean results to Origin. I used the Boltzmann model of the Origin software to create a plot of the mean area data. Voila! There's the sigmoid fit I wanted:

Sigmoid fit for the 5mM DNA isotherm. (And yes, the demo prints are due to the trial version)

Week 3 in a nutshell

Week 3 was more intensive than the previous two weeks. I lost track of the number of ITC runs that I performed over the last week.

I started week 3 by performing an ITC run with the same 5 mM DNA-NaCl solution from last week to check for experimental inconsistencies. The results displayed on the screen after 1.5 hours of continuous ITC humming produced well-consistent data. In order to stretch the plots between the binding and condensation phases even further, I created an 8 mM DNA-NaCl sample and ran it by the ITC machine. The integrated heat peak for the binding phase had been successfully stretched further after this ITC run, resulting in a larger number of plots between the phases. The experimental parameters were very similar in the two runs that I performed using the 8mM DNA-NaCl solution.

Furthermore, I also performed a second heat of dilution for the 6mM Cobalt Hexammine solution in order to account for one anomalous point in the first run. Subsequently, I subtracted this data from the other DNA-Cobalt Hexammine reactions in order to standardize the binding enthalpies involved in those reactions. This was week 3 inside the lab in a nutshell.

Outside the lab, we had an amazing time at the Tuesday brown-bag lunch. Everybody involved with the X-Sig summer research program on campus took part and we had our fair share of laughter and discussion there. On Wednesday, we had a dinner meet at the Quarry Pavilion. The food was amazing! I had a lot of fun outside work during this week.

The rest of week # 4 Preparation for new sample of chicken blood

The rest of the week I kept washing the chicken blood with KTM and Triton X-100 in order to wash away the fat surrounding the cell. In order to wash the blood, I used 20 micro-liters of KTM and 70  micro-liters of Triton X-100 for each sample and spin it in the centrifuge with a force of 3600xg for 10 minutes at 4 degrees Celsius. Throughout the day I just kept washing the chicken blood and made more KTM because I was running out. This process kept on going from Thursday to Friday because I still had red solution in my sample which is not what I was looking for. This process continued until I can obtain white/clean nuclei. On Friday I was able to see results from washing the chicken blood and I could see white/clean nuclei for two of my four samples. I ran the same procedure for the two solutions once again and I was finally able to get white/clean nuclei for all my solutions. Figure #1 shows how the chicken blood must look after all the washes with KTM and Triton X-100.

Figure #1 White clean Nuclei

After this was done, I once again wash the clean nuclei with just KTM in order to get rid of the Triton X-100. I did this procedure twice and by the time I was done the day was over. The washing of the chicken blood takes a good amount of time due to the amount of Triton X-100 that I added but for future reference I would recommend starting with at least 80 to 100 micro-liters of Triton X-100 and go down on the volume from there.

First Days of week #4 Trial and Error

I started out my week by once again doing the Trial Digestion chromatin due to the results we kept on getting from our DNA and Gel test. I started my work where I create samples with proteinase K, which contain 5 micro-liters of digested chromatin that I had previously created, 5 micro-liters of Proteinase K that professor Andresen recently bought, 0.5 micro-liters of SDS and for this new trial I added 0.55 micro-liters of CaC12 to the sample. After all this solutions where added, I let the samples sit for 50 minutes at a temperature of 50 degrees Celsius. While I was waiting for this to be done, I started preparing the gel once again. Once the gel was done, I placed the gel on the refrigerator for about 1 hour and 30 minutes. Figure one depicts the machine use to heat up the samples.

Figure #1 Isotemperature 

Once the 50 minutes were up, I took out the samples and placed them into ice. By this time I got new micro-centrifuge tubes and took 1 micro-liter of solutions from samples and added it to the new test tubes. I also added 9 micro-liters of 60% sucrose and 10 micro-liters of DI water. I did this for each sample that we had, and I also added 1 micro-liter of blue dye in order to see it once we added into the gel. During this time I also made DNA latter which contain about 4 micro-liter of DNA ladder and 10 micro-liter of 60% sucrose. During this time I took out the gel out of the refrigerator but notice that I used the wrong Comb! and this was really bad because due to this I needed to redo the gel and this took about the whole day. Once I was done making the gel I let it sit on the refrigerator though out the night. In my spare time I kept washing the new chicken blood with KTM. Figure # 2 displays the right comb to used for the gel.

Figure # 2 Comb used to make holes in the gel. 

The next day I came back, I took out the gel out of the refrigerator once again and this time I had everything  ready to go so I put my samples in the gel and used a machine to provide voltage in order to move the DNA from one side to another. This process took about 5 hours and in the mean time I kept washing the Chicken blood with KTM. Once I came back from lunch, my gel was ready to be view by a UV machine. My results showed a better improvement on the last trials but It still wasn't good enough for what we were looking for. Now professor Andresen and I decided to redo everything from the beginning but this time we will used the new fresh blood that was bought recently  and see how this results come out and compare them our previous results. We aren't so sure why our results came out the way they came out but we are thinking it might have been with storing our chicken blood in the -80 degrees Celsius refrigerator might have something to do with it. I guess this is part of being a researcher, to keep trying and improving on previous results in order to progress and grow both has a researcher but also has a human being.

Week #3 Results from our chromosomes

Week # 3

I started out my day by making 500ml of 10mM Tris and 1Mm CaCl2 at 4 degrees Celsius with stirring.

Took our solution from previous week and split it into two test tubes to get it ready for the centrifuge. Once both solutions were measured, I spun it at a speed of 5000xg for 5 minutes in the Centrifuge machine located in the Science center.

Once this was done, I removed the supernatant from each of the two test tubes. I removed 10ul of solution from each test tube and added 930ul of DI water to use UV-VIS machine. Calculated A260 and A320 wavelength for each solution and its absorbent at that wavelength. 

 After this was done, professor Andresen used the same techniques with the dialysis bag and dialysis clip to add the supernatant and store it. Once this was done, we placed the dialysis bag with the supernatant inside to the 500ml of Tris and CaCl2 that we created early today, and placed this solutions into the refrigerator for the day.

When this was done, I calculated the grams of chromatin and DNA that we had in both our solutions.

The next things Professor Andresen and myself did was to create the Agarose Gel, which is used to separate varied sizes of chromosomes. When making a solution of agarose and TBE buffer we must be careful to not let it stay still and always mix it around because if we let it stay still, it will get frozen and then we will have to start again. This process approximate took about the entire day and once the gel was done we placed it into the gel cast and put it in the freezer overnight.

The next day when I came back, I calculated the DNA concentration of our Tri-Digestion. Once this was done, I used the information from the UV-Vis information to calculated how much NE stock volume to add to our solutions. I created an excel chart to calculated all the NE stock volume in one go, once this was done I added all the solutions I need to such has 100x Deluded Micrococcal to eat up DNA strands and heated to a temperature of 37.5 degrees Celsius for 55 minutes. 

Figure 1# Machine used to heat up solutions

 When this was done I got the gel ready that we made yesterday and place it in a place where we were going to add our samples. Once we took our solutions from the heater we took out 5ul of chromatin, 5ul of proteinase and .5ul of SDS into new test tubes and once again place this into the heater for 50 degrees Celsius. Once this was done, professor Andresen carefully poor the new solutions into the gel and used a machine name EASYcast BIA which pours electricity into the gel, which has a positive and negative charge and tends to move the DNA from the left to the right since DNA is negatively charge it will tend to move to a positively charge. This process approximately takes about 3 to 4 hours until you see a blue color line reach 5cm in the gel and once this has reach that point we place it into a special UV light that will tell us where the 146 chromosomes are. 

Figure #2 Blue line where it needs to stop once DNA moves to the right due to charges.

Figure #3 Shows the line where you must fill gel with TBE buffer

The results we got were not the ones we were expecting so I came back to the laboratory and prepared to do the gel again and solutions to continue the next day.

When I came back the next day, I went to a training section to use a machine called UNICORN which took about half the day which I wasn’t expecting. This machine is going to be used to complete many task and one of them will be to use by our lab group. After this was done, I finish my day off by spinning more chicken blood in the centrifuge and creating gel once again for another try. I prepare everything to do the gel test once again for the next day and that’s all that my day consisted off.

The next day came and we did the same procedure with the Agarose gel, placed the solutions on the gel and used once again 100volts to move the DNA to the other side of the gel, and waited out for about 4 to 5 hours until a blue slash line reached the 5-cm mark. Once this was done we put it into a UV light to see how our results come out, unfortunately our results were not what we expected once again because we couldn’t see the separation of chromosomes. This can be seen in figure #4. 

Figure #4 Results from UV light and our chromosomes

This took approximately half the day and the rest of the day I just continue to spin down chicken blood with KTM in the centrifuge. Next week we are replacing our Proteinase K during our Trig-Digestion because we believe this is changing how our results come out. We will due the same procedure and double check on what could had cause our results. 

End of Week three!

Since my last blog post I have not been able to move forward from the equilibrium dialysis step. I have restarted back to square one twice since then. However, we are continuing to struggle with this step. On the last run we got one of the four samples to actually provide sufficient data so currently I am trying to run eight samples in order to get more sufficient data. I've also been heavily experimenting with running the centrifuge at different speeds to see if that could help optimize the data. I am also eternally grateful for professor Andresen setting up his centrifuge machine in our lab. This saves me from sprinting back and forth from the science center and Masters Hall a hundred times a day. When we switched machines however, I had to figure out the RCF (relative centrifugal force). Where two machines at the same rpm can provide varying forces on the things inside, RCF is constant between everything. So after switching machines I upped the RCF by about 1.5x per run, in order to see if this would yield better data. I could then run the supernatant or sample in  UV-Vis machine to see where both the gold and DNA was and had gone too. This allowed us to conclude that spinning at 3900 RCF was the best option for us right now. With my current eight samples that I'm using, I initially spun all eight of them, but spun four of them an additional time before taking the supernatant off and re hydrating. When we finally get data this should allow us to see which method works better. I have another spin or two to finish by the end of today but I am hoping to start Monday off with getting some great data!

Week #2 Preparation of Mononucleosomes

I started my week by getting my hands in chicken blood that I was going to used to extract its DNA and prepare mononucleosomes. The amount of chicken blood that I got from a refrigerator at -80 degrees Celsius  was about 50 mL of blood solution and 25 mL of blood cell volume. Then I added KTM with PMSF (Used to keep proteins from destroying the cell)  already added in the KTM to have a total of 100 mL of blood and KTM, after this was done I split the solution inot 4 x 25 mL solutions. This can be seen in figure #1.

Figure #1 Chicken blood and KTM

After these solutions where made, I had to measure their weight and make sure all of them weight the same mass, this needs to be done in order to used the Centrifuge machine. The centrifuge machine works by spinning solutions at high speeds which makes the gravity increased around the solution and helps separate liquids that have different weights. For our solutions, this process separates blood cells from plasma cells. Since we separated our solution into four different blood samples we only needed to get he same weight for each of them and placed two solutions on each side in order for the solutions to be balanced in the Centrifuge. If for some case we only have one solution, we need to used water has our other sample in order to used the Centrifuge and have a balance sample. Once we spin our sample in the Centrifuge at 3900xg for 5 minutes and then removed the supernatant carefully. The supernatant is the liquid that is on top of the nuclei which is shown in figure #2.

Figure # 2 Supernatant after spin

I did this procedure four times, and after this was done I added 0.2% of Triton x-100, which is a substance used to wash away the fat surrounding the cell. Then we spin the solutions in the Centrifuge at a speed of 3600xg for 10 minutes at a temperature of 4 degrees Celsius. When this was done we incubate for 30 minutes on ice while turning the tubes every 5 minutes. This process took the whole day, and so after all of this was done we placed it in the refrigerator and came back to it the next day.

The next day came and I did the same process has the previous day, I measure the mass of the four solutions we had and added Triton x-100 to wash our solution once again. I repeated this same procedure until I obtain white/clean nuclei. This can be seen in figure #3.

Figure #3 White/Clean nuclei

 One I got this results, I added the four solutions into two test tubes and removed all supernatant. On this two test tubes I added KTM which includes PMSF in order to wash and get rid of the Triton x-100, and I did this twice. When using the Centrifuge, I set it to 3600xg for 10 minutes each time. When the washing was done, I added 8mL of ML and spin it at 3000xg for 5 minutes at a temperature of 4 degrees Celsius. I did this process four times and each time I measured the mass of each test tube in order to use the Centrifuge. This process once again took the whole day, so after I was done with this, I placed the solutions into the refrigerator.

The next day I took 2 small samples of the solution and used the UV-VIS in order to determined the DNA concentration, we used the wavelength of 260 and 320 and determined its absorbent.  In the beginning I was having trouble with the UV-VIS because of issues with the computer software, the problem was solve by restarting the computer once again. This procedure took about two days just because myself and Professor Andresen weren't sure if the data was correct. Once again we did this procedure by taking 10uL of nuclei from our solutions and then adding 930uL of H2O and then adding 50uL of 2M NaOH and 10uL of SDS. Our results can be seen in figure #4.

Figure #4 

Once we got this results, I re-suspended the nuclei in 15mL of ML and PMSF, and split it into 7mL in each 15 mL tube. This solutions were then spun at 3000xg for five minutes at 4 degrees celcius and discarded the supernatant once again. This was done three more times.  When this was done I needed to bring our solution to a temperature of 37 degrees Celsius. Since our nuclei was about 18 to 19 mL, I needed to split it into 4.5 15mL tubes in order to fit it into the Iso-temperature machine, and this was left in the machine for 10 minutes. Once the 10 minutes were off, professor Andresen added 8.3uL of Micrococcal nuclease and we let it seat in the machine for 30 minutes. After the 30 minutes, I added 368uL of EDTA in order to stop the reaction of cutting the DNA. I ice it for 10 minutes and then pour the solution into one test tube, proceeded by placing it into the centrifuged for 5 minutes at a speed of 1000xg at 4 degrees Celsius. I took out the solution from the centrifuge once it was done and I removed the supernatant and kept it. I also made 500mL of EDTA with a concentration of .250uL of EDTA and the rest was water. The next part was done by Professor Andresen because it takes a lot of skills and precision to get it right on the first try. He used Dialysis clip and Dialysis tubing to create a sort of bad for the nuclei to rest at. The dialysis bag is used to let liquid through but not anything else, once this bags were created, they were placed into the 500mL of EDTA and left overnight on the refrigerator.
Friday I started my day by getting training in laboratory safety procedures and then once this was done I headed to the lab. Once I got into the lab I took out our solutions from the refrigerator and from the EDTA that was placed in and pour it into two tubes. I took out a small sample from each solution to see how much DNA concentration we had and we did this by using the UV-VIS has previously done. Once the concentration was found we put our solutions into a stronger centrifuge in order to spin it to a speed of 8000xg for 20 minutes in order to spin down the foggy membranes and debris. When this was done, we should had 80-90% of the total post-digestion DNA in the supernatant and the other percentage in the pellet. I combined the two supernatant from both solutions into one tube and added concentrated stock to make 50mM of NaCl. We placed our solution into a beaker with a spinner and left it stir it slowly at 4 degrees Celsius for the weekend. We will check back at our results in the next week.

Week 2- More ITC stuff

For those of you wondering, here's what the TA Nano-ITC instrument really looks like!

I had a disastrous start to week 2. On Monday, I was in bed for most of the day with a flu. However, things started to pick up on Tuesday. I started off by running the ITC machine on the DNA sample that I made last week. My job was to make the 3mM DNA-NaCl solution react with the 6mM Cobalt Hexammine solution(also made last week) and record the enthalpy changes that signify the two binding phases of DNA, i.e. the binding of DNA with Cobalt Hexammine and the subsequent condensation of the DNA molecule.

I started my experiment by loading the Nano ITC device with the reactants. I loaded the sample cell of the ITC machine with 300 µl of the DNA solution. Next, I loaded the buret syringe with 50 µl Cobalt Hexammine. The process of loading the buret syringe was extremely difficult and stressful the first time. I had to make sure that there were no air bubbles present in the Cobalt Hexammine solution contained in the syringe column. Not only that, I had to poke in a air bubble at the top of the liquid column using the plunger. I remember sweating profusely while desperately trying to pipette air bubbles out of the column! That was a time consuming process.

The next part was pretty straightforward.

 I just used the ITCRun software installed in the computer connected to the ITC device to operate the machine. I basically set all the parameters required for the reaction to proceed and the software did the rest. After equilibrating the sample solution for about 30 minutes, the ITC started emptying the Cobalt Hexammine,bit by bit, into the sample cell after regular intervals of 175 s. At the same time, the software plotted and recorded enthalpy peaks after every 175 s. 

Sadly, my first trial was a massive fail with the first few plots going horribly wrong. I'm assuming it was due to the presence of a massive air bubble at the tip of the syringe. 

Results from run 1

I re-ran the experiment with the same settings for a second time. Didn't work this time either!
When you fail, you have to try harder. That's what science is all about.
So, I reset the experiment for a third time and started the experiment with fingers crossed. And it worked this time!

Result from run 3

I re-modeled the raw data using the NanoAnalyze software to display the thermodynamic parameters involved with the experiment. This was necessary to prove the presence of two distinct binding phases of DNA. 

Remodeled data from run 3

In order to ensure consistency in the data I ran the experiment once more. The results from this run looked similar to the results from the third. Hence, this run was successful too!

Our next objective was to create a broader spectrum of peaks in between the two binding phases of the DNA. In other words, I had to figure out a way to shift the sigmoid curve towards the right and magnify it. I used the simulation mode in the NanoAnalyze software to devise parameters that would enable us to meet this requirement. I won't go into details about how this simulation mode works. But here is a model that the simulation mode prepared for us:

 It recommended that I use a 5mM DNA solution for my next ITC run. I made a 5mM DNA-NaCl solution by dissolving 16.7 mg of calf thymus DNA in 10 ml NaCl. 

I ran this new DNA solution in the ITC with the 6mM default Cobalt Hexammine solution. Here are the results so far:

Results from 5mM DNA w 6mM Cobalt Hexamine run 1

The raw heat data displays a wider range of peaks between the binding phases. Hence it is safe to say that the experiment was successful. To confirm this, I remodeled the raw data using NanoAnalyze:

A perfect match with the experimental model from the simulation mode! 

This week has been an eventful one. Starting off with a few bad trials, I was eventually able to make things work and obtain much better results. A great end to week 2!

Week #2 MTW

So like I last mentioned, I did start off Monday by characterizing the DNA in Professor Thompson's UV-Vis machine. The peak wavelength was at 527nm, this is a good sign. A solution with this peak wavelength indicates that the majority of the gold nano-particles are the size and shape that we want them to be. I then used a quartz cuvette in the UV-Vis machine to examine the DNA. The peak was then at 258nm which is a good place for the DNA's peak to be. We also recorded the wavelengths at 260nm and 320nm. This was done because subtracting the 320 wavelength from the 260 wavelength can allow us to calculate the concentration of the DNA in the solution.

Next I began preparing for equilibrium dialysis. First I prepared some TEM buffer, which is like TE buffer but slightly different. It contains NaCl, Tris, EDTA, and water. This would be what is used to re-hydrate the solutions during dialysis. Then I did more calculations to find the proper ratio to mix the DNA and gold nano-particles together. Unfortunately (?) something must have gone awry, for the DNA and gold nano-particle solutions (which all SHOULD have contained exactly the same thing) looked very different.


It's very clear that these solutions are not all the same. We decided to re-run these samples in the UV-Vis to try to identify where things had gone wrong. The UV-Vis showed us that the samples were all relatively the same, so we continued to equilibrium dialysis. For equilibrium dialysis we use big centrifuges located in Professor Thompson's lab. The procedure calls for 40 minutes at 3000rpm. Unfortunately, I ran the first 40 minutes at 3000rpx (?) which is roughly 5000rpm. This is much more forcible then what should be used, and likely caused the DNA to crash out. After the first spin it looked like this:


To me, this looked pretty normal. The pellet is not very big, but is definitely noticeable. The supernatant was then siphoned off and the solution was rehydrated with TEM buffer. The second run, I was sure to set it at 3000rpm for 40 minutes. After the second run the solution looked like this:


It is very noticeable how small the pellets are and this concerned me. I then siphoned off the supernatant and re-hydrated the solution with TEM buffer and ran the solution at 3000rpm for 40 minutes in the centrifuge one last time. After this last run, the pellet was almost unnoticeable. Regardless, I siphoned off the supernatant and re-hydrated the solution. Because the pellets were so small on the second and third runs, we figured that something was wrong. To try to find what had gone wrong, we decided to run everything in the UV-Vis machine again. After getting the results back, it appears that the first run at 3000rxm had in fact caused the DNA to crash out.

Week #1

Introduction to Lab requirements and goal

The first week working with Dr. Andresen was a great success and great experience. We started our Research with a basic understanding of what our goal was and proper ways to work in an environment where all data must be collected and solutions must be carefully made.

Monday, May 15, 2017

On Monday I met with Professor Andresen and his summer research group to discussed the plan and the goal for the research that we are investigating. We went over the basic requirements that we need to accomplish such getting keys to lab room so we can have access when Professor Andresen can't make it. We also went over the websites that our group must used in order to keep track of our progress and find preparation guides for solutions that we might need in the near future. This website is called lab wiki and we can find useful information on how to specifically do a solution and it also gives us the freedom to create our own page where we can post a guide on the techniques that we used to create a specific solution. I was given Abby's and Sarah's work book to gather information on their procedures and work done previous summer's. I was also given articles and books related to the cell and specially Nucleosome Core Particle to learn more about how the cell works and the role of DNA.

Tuesday, May 16, 2017

On Tuesday I made 50ml of 1 Mole stock of Na-Cl which is about 2.922g. After creating this solution I added the Na-Cl into a disposable container  of 50mL and label it with the name of the solution, my initials and the day it was created. This was also done to 1 M of Magnesium Chloride hexahdrate and I used 10.165g of stock  creating 50ml. After creating this solution I went to the wiki page and look for the preparation of Tris-HCl. Printed our the instructions and taped them into my workbook, and I made the solution following each step with the help of Professor Andresen, since I was dealing with powerful acid that I wasn't used to work with. This was done through out the day, seeing how I was learning how to used the proper techniques and tools to complete each task, this was all done with the help and guidance of Professor Andresen.

Wednesday, May 17, 2017

I started out my day by creating 50ml of EDTA with about 1/2 molar, this translate to 9.3004g of stock. On this day Professor Andresen added NaOH to get the content to an pH of 8.0. While the Professor was doing this, I was left the task to make 500ml of KTM, Ml, and DB solutions. Each solution contain different components such has Tris-HCl, NaCl, and MgCl2. The procedure in making this solutions can be found in documents provided by previous research members. This solutions and data took the entire day, careful measurements were made and tools used were washed to keep a clean laboratory and environment.

Thursday, May 18, 2017

I was left with the task to make more Tris-HCl with pH of 7.5. From what I learned from Professor Andresen, I took careful measurements of the amount of HCl I added to the Tris-HCl in order to control the pH to be 7.5. I also was very careful in handling HCl, by wearing protective gloves and plastic glasses. After creating 50ml of Tris-HCl, I added the procedure to make EDTA onto wikipedia. After this was done I helped Dylan when using the UV-VIS machine, but we encounter some software problems that created problems in our solutions for the nano-particles. The time I had between projects, I read over all the documents that I still had from Professor Andresen, and read more about the structure of DNA in a cell.

Friday, May 19, 2017 

On this day we started by having a group meeting with Professor Andresen and my colleagues. This group meeting was to inform Professor Andresen about our progress in our work and an overview of what we had done during the week. On this day we also got information on software that will be helpful in future research of scientific articles and citations. When this was one, my colleagues and I went to the Science Center to shred DNA and we took careful notes on every step we did. When this was done we launch the DLS machine and got results from the nano-particles with used. During this time we also got more nano-particle from last summer that Savana was using, and to finish the day off I spend relocating the nano-particles into new containers and reading useful articles that Professor Andresen provided, in order to prepare for next week.

Week 1- Isothermal Titration Calorimetry

The first week of my research with Dr. Andresen started with some detailed study about the Nano Isothermal Titration Calorimetry (ITC) technology. The basic idea of our research project is to figure out the thermodynamics associated with DNA condensation by Cobalt (III) hexamine binding. Fundamentally, we are trying to reproduce the results from another similar experiment that deals with the enthalpy change of DNA condensation.

Day 1: Monday, 15th May, 2017

I started out by watching youtube videos on how the Nano ITC machine works. ITC is a technique that deals with a wide variety of bimolecular interactions. It directly measures hear either released or absorbed during a biomolecular binding event and it is extremely sensitive to very small heat changes. The Nano ITC machine consists of two identical cells, made of gold due to its inertness and high thermal conductivity. These cells are surrounded by an adiabatic jacket. The instrument uses two very sensitive thermocouples, one in each cell to constantly monitor the temperatures of the cells. The temperatures of the cells are kept equal. The heat supplied to the sample cell will be lass than the heat supplied to the reference cell if the reaction is exothermic and vice versa.

Day 2: Wednesday, 17th May, 2017

Wednesday was all about synthesizing the chemicals required for measuring the heat of dilution using the ITC machine. We were to measure the heat evolving from the dilution of cobalt hexamine using 10 mM NaCl solution. I started by making a 0.2 M Cobalt Hexamine stock solution. I then used the 0.2 M stock solution to make 10 ml of 6mM Cobalt Hexamine solution. Subsequently, I made 10 ml of 10 mM NaCl solution from a 1 M stock by dilution with water. I split the 10 ml solution into two tubes, one with 2 ml of NaCl and the other with 8 ml of NaCl (for the DNA solution).

Day 3: Thursday, 18th May, 2017

On Thursday, I used the Nano ITC machine to measure the enthalpy of dilution of cobalt hexamine. I had to be extremely careful while loading the syringe with 50 microliters 6mM Cobalt Hexamine, to make sure that there were no air bubbles inside. Fontaine helped me with loading the reference cell with 300 microliters of water and the sample cell with 300 microliters of  10mM NaCl solution. I had to be extremely cautious so that there were no air bubbles inside the cells. The presence of air bubbles in cells or the syringe can give erroneous results. The injection interval for the cobalt hexamine injection was set to 175 s, 20 injections in total. The ITCRun software was used to operate the instrument. The spin rate was set at 250 rpm and the temperature was held constant at 25 C. The solutions were allowed to auto equilibrate for 1564 s. After that, the syringe automatically started injecting 2.5 microliters of Cobalt hexamine solution after every 175 s intervals. The NanoAnalyze software was used to measure enthalpy peaks after each injection:

As evident from the NanoAnalyze model, the peaks are more or less overtime (except for that last peak which we discarded as an experimental anomaly). The heat evolved after each injection is measured by integrating the area under the curve. The normalized fit for integrated heat vs number of injections was also plotted using NanoAnalyze. Turns out, the normalized fit is not exactly sigmoid as we would expect it to be.

Day 4: Friday, 19th May, 2017

After the satisfactory results from our first test run of the ITC instrument, we decided to start making the DNA-NaCl solution with a concentration of 1 mg/ml. Dylan guided me through the processes of measuring out 8 mg of DNA using the analytical balance, dissolving the DNA in 8 ml of 10 mM NaCl solution and subsequently setting up the solution for DNA shearing. After we completed the process of DNA shearing, we refrigerated the resulting solution. A few hours later, I checked the pH of the solution for consistency. I measured the pH to be somewhere between 6 and 7. The next step would be to measure the enthalpy change of DNA condensation after it reacts with Cobalt Hexamine in the Nano ITC instrument (to be done on Monday). Earlier that day, Dylan also introduced me and Jose to the UV-Vis spectrophotometer and he showed us how to operate the device. Dylan also made demonstrations on how to operate the DLS machine. 

Week #1

We picked up this summer almost exactly where I left off my training with Savannah. Since I first started, the organization of the lab had been bothering me. Especially the bookshelf, I just did found the entropy to be too high. This is no longer the case! A very thorough cleaning has left us with several empty drawers, an alphabetized bookcase and an alphabetized cabinet of  samples. Additionally, I prepared all of the samples required to calibrate the ICP and the calculations required to do so. This process included massing several solutions and then diluting them to a specific concentrations. After I had done this Professor Andresen believed that I was qualified enough to write my very first Wiki article on the subject. The process of creating my very first wiki page was exhilarating, it feels almost like a child to me now. Today, Wednesday, I was almost able to set up the ICP machine by myself. My goal for today is to run the ICP on the samples that I prepared yesterday. Below is a picture from my notebook of the theoretically calculated masses of the samples I prepared and below the theoretical masses are the actual ones.

Thursday I worked with Jose and he taught me how to prepare the TRIS-HCl solution that I had used previously in my TE buffer. Later I taught him how to use the UV-Vis machine, although we ran into some troubles with it. Apparently the machine was attempting to connect to its thermal regulator however, there was non connected to the machine. Using this machine should of taken less then a half hour but through a series of mishaps it ended up taking at least an hour and a half with some professor guidance. I also helped Jose learn how to shear DNA. Originally we were doing this in Professor Frey's lab, however she has since moved the Sonic probe and the DLS machine to the Pchem laboratory. This was a pretty good move to get this out of her office space, as the noise the probe makes is one of the most unpleasant things I've ever heard. 

Friday we kicked it off with a pretty productive group meeting that left everyone with a nice list of things that they could work on. My tasks included using the UV-Vis machine, helping Amlan shear DNA, using the DLS and Zeta machine. Towards the end of the day it was looking like I was going to accomplish everything until I found that my saved file on the UV-Vis machine was lost. Additionally, another student was now using the UV-Vis machine in Professor Thompson's lab. I then returned to Professor Andresen's lab to use the UV-Vis machine there. Unfortunately we were once again accosted with technological mishaps. We threw in the towel and decided to deal with UV-Vis on Monday morning. I ended my first week reading a very helpful research paper outside in front of the fountain.