Getting the Stain IN

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Getting the Stain IN

• A Quantitative Analysis of DNA and RNA Staining
  with Various Dyes
• Dr. Amber Charlebois
• Teaching Assistant Jeremy Tang

Rupak Bhuyan Lauren Dai Tyler Davenport Bruce
Easop Arka Mallela Kenneth Pu Shireen Rudina Tina
Sankhla Margaret Yang
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Purpose

• To identify dyes that
• Can differentiate between DNA and RNA in gel
  electrophoresis
• Are safe, inexpensive, and effective
• Have a linear relationship between amount of
  nucleic acid and absorbance

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Project Overview

• Nucleic Acid Negatively Charged
• Polyacrylamide Gel Electrophoresis (PAGE)

http//en.wikipedia.org/rna
(2007). Retrieved July 29, 2008, from Southern
Illinois University School of Medicine Web site
http//web.siumed.edu/bbartholomew/images/chapter
6/F06-21.jpg
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(Begin Run)
Loading Dye
Loading Dye
Loading Dye
RNA
DNA
RNA
DNA
RNA
Loading Dye
RNA
DNA
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(destain in water)
Staining the Gel
DNA band 35 bases
RNA band (18 bases)
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Use of Fiber Optic Technology
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Use of Fiber Optic Technology
Halogen Source
(2006, September 26). Introduction to the Ocean
Optics Spectrometer System . Retrieved July 29,
2008, from Truman State University Chemistry
Laboratory Web site http//chemlab.truman.edu/Ins
trumentation/OceanOptics/OOIntro.htm
Detector
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Dyes
Cresyl Violet
Methylene Blue
Toluidine Blue O
Azure B
Thionin
Azure A
Azure C
Brilliant Cresyl Blue
Coomassie Brilliant Blue
Nile Blue A
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General Testing of Dyes
Strips of Gel Stained with Methylene Blue, Azure
A, and Azure C prior to destaining
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Azure B
RNA DNA
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Azure A
RNA DNA
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Azure C
RNA DNA
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Toluidine Blue O
RNA DNA
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Cresyl Violet
RNA DNA
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Brilliant Cresyl Blue
RNA DNA
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Methylene Blue
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Thionin
RNA DNA
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Coomassie and Nile Blue A
Coomassie
Nile Blue A
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Dyes Chosen for Quantitative Analysis
Cresyl Violet
Methylene Blue
Toluidine Blue O
Azure B
Thionin
Azure A
Azure C
Brilliant Cresyl Blue
Coomassie Brilliant Blue
Nile Blue A
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Thionin
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Azure B
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Cresyl Violet
DNA
RNA
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Methylene Blue
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Quantitative Data Azure B DNA
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Quantitative Data Methylene Blue DNA
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Quantitative Data Cresyl Violet DNA
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Quantitative Data Thionin DNA
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Corrected Thionin DNA Data
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Quantitative Data Azure B RNA
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Corrected Azure B RNA Data
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Quantitative Data Cresyl Violet RNA
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Quantitative Data Thionin RNA
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Quantitative Data Methylene Blue RNA
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Corrected Methylene Blue RNA Data
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Conclusions

• Methylene Blue metachromatic, linear for RNA up
  to 100 picomoles
• Azure B shows promise for linearity for RNA
• Thionin abnormal pattern for DNA
• Cresyl Violet needs further research
• All other dyes except Nile Blue and Coomassie
  show promise in nucleic acid staining

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Future Research

• More careful pipetting
• The effect of dye band area on absorbance values
• Mixing Dyes
• Use of other statistical methods to determine
  linearity
• Multi-linear regression pattern

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Acknowledgements

• Dr. Miyamoto
• Dr. Paul Quinn
• Counselors
• Schering-Plough Foundation
• Novartis
• The Dorr Foundation
• The Jennifer A. Chalsty Foundation
• The Edward W. and Stella C. Van Houten Memorial
  Fund
• The Jewish Communal Fund
• Laura and John Overdeck
• NJGSS Alumni and Parents 1984-2008

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Questions?