Difference between revisions of "Team:CUNY Kingsborough/Description"
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<h2 class="default-padding" id="E">Ethidium Bromide Spot Test</h2> | <h2 class="default-padding" id="E">Ethidium Bromide Spot Test</h2> | ||
| − | <p class=" | + | <p class="low-rise-padding">Quantifying DNA may be time – consuming, especially if results are not as expected. One protocol that may help speed up the process of DNA quantification is the Ethidium Bromide Spot technique. The protocol, developed recently, requires a minimal amount of EtBr and DNA to measure DNA concentration. It also decreases contact time with harmful EtBr and does not require the use of a spectrophotometer or nanodrop machine. Last year, our team collected data from the images of diluted DNA in EtBr to create a standard curve varying pixel intensity of the DNA sample and its concentration. This year, we are improving our standard curve by finding its accuracy and adding more data – in hopes of measuring DNA as accurately as a spectrophotometer or nanodrop machine may. We hope to further standardize the EtBr Spot protocol so future beginner or funding-limited researchers and teams working with DNA can accurately perform their experiments.</p> |
Revision as of 05:44, 17 October 2018
Description
Background
JULIAN REWRITE THIS
One of the challenges in synthetic biology is characterizing the random nature of most synthetic biological systems. Where engineered physical systems can be predicted accurately, similar attempts to predict biologically engineered systems as accurately as physical systems have been a challenge. However, not enough research has been done to study how to harness the natural randomness of biological systems. For instance, the waste treatment industry spends a significant amount of energy in maintaining biosystems for waste treatment. Therefore, creating a stable biosystem is desirable for industrial purposes. The reaction-diffusion system, first proposed by Alan Turing, offers an attractive solution. Using the random diffusion of activators and inhibitors-it is worth exploring how to harness the randomness of this system to create a stable biosystem.
Light Operon
Ethidium Bromide Spot Test
Quantifying DNA may be time – consuming, especially if results are not as expected. One protocol that may help speed up the process of DNA quantification is the Ethidium Bromide Spot technique. The protocol, developed recently, requires a minimal amount of EtBr and DNA to measure DNA concentration. It also decreases contact time with harmful EtBr and does not require the use of a spectrophotometer or nanodrop machine. Last year, our team collected data from the images of diluted DNA in EtBr to create a standard curve varying pixel intensity of the DNA sample and its concentration. This year, we are improving our standard curve by finding its accuracy and adding more data – in hopes of measuring DNA as accurately as a spectrophotometer or nanodrop machine may. We hope to further standardize the EtBr Spot protocol so future beginner or funding-limited researchers and teams working with DNA can accurately perform their experiments.
How is DNA Quantified?
DNA can be quantified through gel electrophoresis, a process that separates proteins in a sample by charge and molecular weight - with the lighter proteins traveling further down a gel and the heavier ones staying on the top. Since DNA is negatively charged, the more nucleotides in a sample (meaning the more DNA) the slower it will migrate to the end of the gel. These proteins are seen as bands on the gel - however, to truly visualize them the gel must be dyed with an agent such as EtBr.
How does EtBr Work?
Ethidium Bromide is an intercalating agent - this means that it inserts itself between the nucleotides of a nucleic acid such as DNA or RNA. It has been shown that the amount of EtBr intercalating throughout a sample is proportional to its concentration.
Once the agarose gel is stained with EtBr, it is run and imaged. During imaging, the gel is hit with UV light to visualize the bands. Fluorescence occurs because EtBr is an aromatic compound, meaning it contains many double bonds. When EtBr is hit with UV light, these double bonds absorb energy from the visible light at a certain wavelength and reflect light at others. The orange color we commonly associate with EtBr is the result of reflected light of a particular wavelength.
