iGEM 2018 — Team CUNY_Kingsborough

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# Team:CUNY Kingsborough/Notebook

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[HOME](https://2018.igem.org/Team%3ACUNY_Kingsborough)

TEAM

[Team Members](https://2018.igem.org/Team%3ACUNY_Kingsborough/Team)
[Collaborations](https://2018.igem.org/Team%3ACUNY_Kingsborough/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3ACUNY_Kingsborough/Description)
[Design](https://2018.igem.org/Team%3ACUNY_Kingsborough/Design)
[Experiments](https://2018.igem.org/Team%3ACUNY_Kingsborough/Experiments)
[Notebook](https://2018.igem.org/Team%3ACUNY_Kingsborough/Notebook)
[InterLab](https://2018.igem.org/Team%3ACUNY_Kingsborough/InterLab)
[Model](https://2018.igem.org/Team%3ACUNY_Kingsborough/Model)
[Results](https://2018.igem.org/Team%3ACUNY_Kingsborough/Results)
[Demonstrate](https://2018.igem.org/Team%3ACUNY_Kingsborough/Demonstrate)
[Improve](https://2018.igem.org/Team%3ACUNY_Kingsborough/Improve)
[Attributions](https://2018.igem.org/Team%3ACUNY_Kingsborough/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3ACUNY_Kingsborough/Parts)
[Basic Parts](https://2018.igem.org/Team%3ACUNY_Kingsborough/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3ACUNY_Kingsborough/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3ACUNY_Kingsborough/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3ACUNY_Kingsborough/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3ACUNY_Kingsborough/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ACUNY_Kingsborough/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3ACUNY_Kingsborough/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3ACUNY_Kingsborough/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3ACUNY_Kingsborough/Hardware)
[Measurement](https://2018.igem.org/Team%3ACUNY_Kingsborough/Measurement)
[Model](https://2018.igem.org/Team%3ACUNY_Kingsborough/Model)
[Plant](https://2018.igem.org/Team%3ACUNY_Kingsborough/Plant)
[Software](https://2018.igem.org/Team%3ACUNY_Kingsborough/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=CUNY_Kingsborough)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:CUNY Kingsborough/Experiments

Project

[Background](https://2018.igem.org/Team%3ACUNY_Kingsborough/Description)
[Design](https://2018.igem.org/Team%3ACUNY_Kingsborough/Design)
[Experiments](https://2018.igem.org/Team%3ACUNY_Kingsborough/Experiments)
[Results](https://2018.igem.org/Team%3ACUNY_Kingsborough/Results)

Modeling

[Overview](https://2018.igem.org/Team%3ACUNY_Kingsborough/Model)
[Modeling 101](https://2018.igem.org/Team%3ACUNY_Kingsborough/Modeling_101)
[Light Operon](https://2018.igem.org/Team%3ACUNY_Kingsborough/Light_Operon)
[Turing Patterns](https://2018.igem.org/Team%3ACUNY_Kingsborough/Turing_Patterns)
[CRISPR-Cas13a](https://2018.igem.org/Team%3ACUNY_Kingsborough/CRISPR-Cas13a)

[![](https://static.igem.org/mediawiki/2018/0/0e/T--CUNY_Kingsborough--2018Logo.jpeg)](https://2018.igem.org/Team%3ACUNY_Kingsborough)

Human Practices

[Human Practices](https://2018.igem.org/Team%3ACUNY_Kingsborough/Human_Practices)
[Public Engagement](https://2018.igem.org/Team%3ACUNY_Kingsborough/Public_Engagement)

Team

[Team Members](https://2018.igem.org/Team%3ACUNY_Kingsborough/Team)
[Collaborations](https://2018.igem.org/Team%3ACUNY_Kingsborough/Collaborations)
[Attributions](https://2018.igem.org/Team%3ACUNY_Kingsborough/Attributions)

# Experiments

## Ethidium Bromide Spot Test

Accurate and quick determination of DNA concentration is critical for the assembly of synthetic constructs, as well as a multitude of other experiments. We sought to optimize an under-utilized and inexpensive approach for determining DNA concentration: a spotting technique that uses the intercalating dye Ethidium Bromide. This technique does not require specialized equipment such as a spectrophotometer, but instead relies on visualization of dye-DNA complex fluorescence when excited by UV light. We modelled and tested a range of parameters for dye concentration and spot size, finding that 15uL spots with 1.0ug/mL Ethidium Bromide produced the most reliable standard curve. A detailed paper with our findings from 2017 data can be found here: [Quantification of DNA samples by Ethidium Bromide Spot Technique](https://www.biorxiv.org/content/early/2018/03/27/289108).

## Protocol

Below is the procedure used for data collection in 2017 and what we sent to iGEM teams in 2018 as part of [Collaboration](https://2018.igem.org/Team%3ACUNY_Kingsborough/Collaborations):

5-15uL of diluted Ethidium Bromide was pipetted onto a disposable plastic petri dish. 1uL of water or diluted lambda DNA was then pipetted into the droplet/spot containing the dye and mixed. The dish was then inverted over a UV transilluminator. A cover shielded ambient light and a photograph was taken using an orange light filter and an android smartphone (Motorola Droid 2).

## Image Processing

Images of the spots were processed using ImageJ®. Briefly, spots were selected using a freehand or oval tool, either to analyze the entire spot(Whole Area) or the region within the spot with the brightest uniform intensity (Highest Fluorescence). Pixel intensity was measured using the analysis tool, with or without a de-speckling correction applied first (Noise reduction). These measurements were either taken as a ratio to a background signal and/or normalized to the maximum value for a set, and averaged across multiple experiments.

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:CUNY Kingsborough/Results

Project

[Background](https://2018.igem.org/Team%3ACUNY_Kingsborough/Description)
[Design](https://2018.igem.org/Team%3ACUNY_Kingsborough/Design)
[Experiments](https://2018.igem.org/Team%3ACUNY_Kingsborough/Experiments)
[Results](https://2018.igem.org/Team%3ACUNY_Kingsborough/Results)

Modeling

[Overview](https://2018.igem.org/Team%3ACUNY_Kingsborough/Model)
[Modeling 101](https://2018.igem.org/Team%3ACUNY_Kingsborough/Modeling_101)
[Light Operon](https://2018.igem.org/Team%3ACUNY_Kingsborough/Light_Operon)
[Turing Patterns](https://2018.igem.org/Team%3ACUNY_Kingsborough/Turing_Patterns)
[CRISPR-Cas13a](https://2018.igem.org/Team%3ACUNY_Kingsborough/CRISPR-Cas13a)

[![](https://static.igem.org/mediawiki/2018/0/0e/T--CUNY_Kingsborough--2018Logo.jpeg)](https://2018.igem.org/Team%3ACUNY_Kingsborough)

Human Practices

[Human Practices](https://2018.igem.org/Team%3ACUNY_Kingsborough/Human_Practices)
[Public Engagement](https://2018.igem.org/Team%3ACUNY_Kingsborough/Public_Engagement)

Team

[Team Members](https://2018.igem.org/Team%3ACUNY_Kingsborough/Team)
[Collaborations](https://2018.igem.org/Team%3ACUNY_Kingsborough/Collaborations)
[Attributions](https://2018.igem.org/Team%3ACUNY_Kingsborough/Attributions)

# Results

## Introduction

To better characterize the standard curve for the EtBr-DNA Dot Blot, we are interested first whether the amount of EtBr significantly affects the fluorescent when the DNA concentration is varied between different amounts of EtBr and demonstrate there is significant difference in fluorescent between. We almost must characterize the predictive ability of the standard curve using some known DNA concentration that is not used for the curve. Kasap et al. (2006) has shown that it is possible to approximate the concentration of DNA using Imagej. In this study we will use a linear regression model to find:

* Is there a measurable pixel difference?
* What are the factors that affect pixel intensity?

## Methods

Statistical analysis was done with R-studio and the Standard Curve was generated using Excel. Mass action model was done using Wolfram Mathematica. Data was entered into Excel and standardized using the following fomula:
StandardPixel = (Value-MIN)\div(MAX-MIN)

## Results

### Predicted Standard Curves ![](https://static.igem.org/mediawiki/2018/7/75/T--CUNY_Kingsborough--iGEMLinearStandardCurve.jpg) We examined the correlation of 125ng DNA and 16ng DNA under varied conditions of EtBr. We found that there is a low correlation when between the amount of EtBr and fluorescent. 125 ng of DNA vs EtBr ![](https://static.igem.org/mediawiki/2018/a/a8/T--CUNY_Kingsborough--iGEM125ngDNAvsEtBr.jpg) Fig 1. Scatter plot of 125ng DNA with varied amount of EtBr. r=0.23 16 ng of DNA vs EtBr ![](https://static.igem.org/mediawiki/2018/c/c7/T--CUNY_Kingsborough--iGEM16ngDNAvsEtBr.jpg) Fig 2. Scatter plot of 16ng DNA with varied amount of EtBr. r=0.20 When number of DNA was varied, we found that 125ng of DNA (β = 43.0, p < .001), and 16 ng of DNA (β=41.9, p < .001) were significant predictors of pixel intensity. Zero ng of DNA was used as the reference variable. The overall model fit was R^2 = 0.57, F-statistics = 41.02, p<.001. This goes in contrast to our Mass-Action model which shows that the amount of EtBr has a significant effect on the pixel intensity. However, it is obvious that EtBr is needed for DNA to fluorescent therefore a future study should examine at what concentration of EtBr do we see no fluorescent when the amount of DNA is varied. ![](https://static.igem.org/mediawiki/2018/6/6a/T--CUNY_Kingsborough--resultsfig1.png) Fig 3. Linear model results of lab data Figure 3 shows the linear model results for our own data. We found that amount of DNA (β=20.99, p<0.05) and EtBr (β=-0.38, p<0.05) are significant factors to pixel intensity. Background is not a significant contributor to pixel intensity. The overall model is significant (R2=0.387, F-statistics = 14.28, p<0.01). ![](https://static.igem.org/mediawiki/2018/9/98/T--CUNY_Kingsborough--resultsfig2.png) Fig 4. Residual plot of figure 3 results Figure 4 shows the residual (Residual = Observed-Predicted). According to the residual, our model seems to under predict observed values and over predict lower values. This suggests that our model has not account for all the possible variables. ![](https://static.igem.org/mediawiki/2018/f/fa/T--CUNY_Kingsborough--resultsfig3.png) Fig 5. Linear model from other teams Figure 5 is the linear model for the DNA-EtBr sample sent by other iGEM teams. DNA concentration (β=5.08, p<0.05) and background (β=-28.38, p<0.05) are significant contributors to pixel intensity. The results in Figure 3 suggests that the background-though we subtracted the background pixel intensity-is still a significant contributor to overall pixel intensity.