Sunday, June 25, 2017

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.

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