Gel Extraction and Transformation

Gel Extraction:

After we have cut and dissolve our DNA from the gel, we can then add ethonal so the DNA falls out and we also add special beads to bind to the DNA so it can separate and stay at the bottom of the test tube. After that we can dissolve the DNA in free water. And we have now successfully extracted our DNA!
                                                     
 DNA can be added to a plasmid or a topovector, which will allow us to insert our DNA into bacteria during the process of transformaiton. This procedure is also called ligation.

Transforamtion:

Transformation is used to "get more DNA." When the plasmid is added to the bacteria, the bacteria grows and the plasmid, containing our targeted DNA, will grow with it.

1) thaw the bacteria
2) add ligation to bacteria
3) incubate ligation and bacteria on ice for 30 min.
4) heat shock > at a higher temp. the bacteria will expand to release heat. And when it is expanded, the plasmid has the ability to travel inside the bacteria. Then the temp. decreases again and the bacteria closes, capturing the plasmid inside.
5) plate the bacteria > the bacteria is given time to grow, and the bacteria with antibiotic resistance will be the only ones left living for our use.

plating the bacteria

bacteria after growth

                                             




Next week we are looking forward to getting the sequencing results, which will give us the neucleotide base sequence of our gene of interest, answering the first part of my project...
Is gene 5 a real gene?
         

February 6, 2015

     Since last week's electrophoresis gel was not very successful, we re-did the procedure and hoped for better results. My mentor crated the solutions again that way I had time to re-imbed the gel with our DNA, and take it down to examine under the UV light.
     Thankfully, our gel worked this time and we were able to see the DNA bands under the UV light. We cut out the bands of DNA and saved them in test tubes, and later I will use them in gel extraction and sequencing further down my project.

     Then after that we went to work on isoform 3. Since these cells were already ready to be put into the cell all we had to do was add lipids to the membrane of the DNA to ensure that the plasmid would be engulfed ny the cell. If the DNA did not have a lipid membran the cell would not take in the DNA that we want it to. With the plasmid we are also going to add an antibiotic resistance and then when we need to we can add antibiotic to our cells and the ones that live are the ones that obsorbed the plasmid with DNA. and those are the cells that will benefit us in our experiment.

Milestone 4

Annotated Bibliography:

• "Roles of RNA in Cells." Umassmed.edu. Accessed December 9, 2014. http://www.umassmed.edu/rti/biology/role-of-rna-in-cells/.

-provides a background to my project

• "Long Noncoding RNA MALAT1 Regulates Endothelial Cell Function and Vessel Growth." Ncbi.nlm.nih.gov. April 25, 2014. Accessed December 9, 2014. http://www.ncbi.nlm.nih.gov/pubmed/24602777.

-also provides a background to my project

• Garbiyan, L., & Acashia, N. (2013). Polymerase Chain Reaction. Journal of Investigative Dermatology. http://www.nature.com/jid/journal/v133/n3/full/jid20131a.html

                -Polymerase Chain Reaction article 

• "DNA Sequencing." Biotechnology Learnign Hub. Newzeland.govt.nz 2007 http://biotechlearn.org.nz/themes/dna_lab/dna_sequencing
  
  

              - DNA sequencing article 

Reflection:

This whole internship experience has been very challenging, but at the same time also very rewarding. Working in a traditional based lab taught me a lot of the basics required, for example, how to pipette or always wear gloves when working with ethidium bromide. But apart from that, the project that i am working on, especially since i am quite new to this whole experience, has required a lot of patience and persistance, not only from me but form my mentor as well. In the beginning it took a little time to absorb and understand the cell's inner chemistry, or why this solution was being added to this tube etc. However once tha information was processed I began working my own portion of the experiment. At this point my mentor trusted me enough to let me carry out my own lab experiments each day I came in. Of course she was there to answer any question that I had. I think that my project is progressing at a steady rate nd hopefully I will be able to keep that pace up with my mentor's help. 

January 16, 2015

Today I sat down with my mentor to speak about my indivisual project that I will be working on. First my project will consist of the question,
     what is the difference between cancerous and non-cancerous cells?
1) to design better treatments.
2) stop pre-cancerous cells before they become harmful.
3) stop treating non invasive breats cancer cells, so we have time to spend on cancerous patience.

Our lab has found a new gene! (gene #5, changed the name for security purposes). We can test this gene by first copying the new gene using RNA extraction, and turning it into DNA to put the DNA with gene #5 into the cell and then observe what happens. But first we need to make sure this wasn't just a fluke. So my job in the lab is to check if this new cell line is an actually exsisting new gene or just an experimental fluke by using the methods of RNA extraction and qPCR.

1. RNA extraction:

• we will have a test tube containing everything from the cell including gene #5. 
• we add primer to attach to only gene #5 
• now we have the gene that we need and to replicate we use polymerase, but we can use RNA poymerase because it dose not work at the temperature of PCR. 
• so first we have to transoform RNA to DNA so we can use DNA polymerase at a lower temperature.  

2. PCR- Polymerase Chain Reaction 

• this process is used to amplify gene #5 at a temperature at 95℃

a) denature 

          b) add primer (short sequence that complements DNA)

          c)  DNA polymerase adds nucleotide bases

Phenol-Chloroform Extraction

     This specific experiment is used to extract mixtures based on their solubilities. In this experiment we used it to extract RNA from DNA and protein. Protein is a non-polar molecule, it will be separated from the DNA which is a polar molecure. The RNA and DNA will end up in the chloroform, which is also polar. And the protein will end up the less polar solution, QIazol. The DNA will separate from RNA as it also moves into QIazol because the negative charge of DNA's backbone will be attracted to the H+ in QIazol. RNA is a single strand, so the exposed nitrogen bases will form hydrogen bonds with chloroform.

      After both solutions were transfered into the test tubes we added them to a centrifuge mixer for 15 min to create the separated layers. Because QIazol has a greater density than chloroform, the QIazol will reside at the bottom of the tube, and will give us easier access to the RNA at the top. Then we can take a pipette and carefully extract the RNA from the test tube.

     We also talked about the role of transcription factors. Transcription factors bind to DNA to make mRNA. DNA is wrapped around a protein histone, and some of it can be unwinded, and on the unwinded DNA there is a start and stop point. There are different stopping places for every different mRNA. And different mRNA's code for different functions and different proteins. There is also something known as "splicing", which means the DNA is cut from the middle, and this creates many message from the same gene.
     The transcription factors can turn on a gene by binding to the DNA, but can also turn off a gene when the gene is not unraveled from the histone leaving there no place for the transcription factor to bind to. Specifically in cancer cells, the transcription factors are always binded to the gene, which is also the reason for the cell's rapid growth. So if the transcription factors can be controled, then the cells would be able to maintain a more stable growth rate.

Capital Region Cancer Research Symposium 2014:

 New Strategies and technologies in Cancer Reasearch

        Today instead of the usual lab work and lab readings, we headed over to the cancer research symposium held at the Hilton inn at Albany Medical center. The purpose of the symposium is to promote cancer research interactions among differnt Albany facilities. There I sat and listened to the different cancer research project all across the capital district. Projects came from SUNY-Albany, RPI, Albany medical center, Albany college of Pharmacy and many more.
        One particular presentation that i found very interesting was "Collective Rotation of Epithelial Cells Exhibits Left-Right Asymmetry in a 3D system," given by Amanda Chin, a graduate student at RPI. Basically she concluded that left-right symmetry in a cell is vital for normal tissue growth. She concluded that inherent cellular chirality (left-right asymmetry) could be a force that moves cells during tissue morphogenesis, and the development of tumors.
        I left a little earlier, the symposium was held until 4:00pm and I left at 11:00am. However I was still able to observe the impressive researches that are happening in our community right now and I got to experience a science symposium, which I had never been to before this.

   

November 7, 2014

Lab meeting

Today during my internship, most of the time was spent in lab meeting; where we went through a paper that was meant to be read the day before. It was quite confusing for me to follow, but after my mentor and I sat down to speak about it, I began to understand the article a lot more. Primarily the article was about the changing of DNA sequence will change the overall function of that DNA, and this can cause "intellectual disabiliy disorder."

Article: NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs

Cell Movies

After the lab meeting, my mentor and I talked about the cell movies, where we looked at the movment of previously cultured breast cancer cells. In the control, we can see that the movement of the cells is not as rapid and agressive as the other cells. Below are movie links to comapare two different cell movements due to different RNA concentrations. 

cell movie: control

cell movie