getting the stain in a quantitative analysis of dna and rna staining with various dyes dr. amber...
TRANSCRIPT
![Page 1: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/1.jpg)
Getting the Stain INA Quantitative Analysis of DNA and RNA Staining with Various Dyes
Dr. Amber Charlebois
Teaching Assistant: Jeremy Tang
Rupak BhuyanLauren DaiTyler Davenport Bruce EasopArka MallelaKenneth PuShireen RudinaTina Sankhla Margaret Yang
![Page 2: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/2.jpg)
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
![Page 3: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/3.jpg)
Project Overview
Nucleic Acid – Negatively Charged
Polyacrylamide Gel Electrophoresis (PAGE)
(2007). Retrieved July 29, 2008, from Southern Illinois University School of Medicine Web site: http://web.siumed.edu/~bbartholomew/images/chapter6/F06-21.jpg
http://en.wikipedia.org/rna
![Page 4: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/4.jpg)
Loading Dye RNA DNA
Loading Dye
Loading Dye RNA
RNADNA
Loading Dye
RNA DNA
(Begin Run)
![Page 5: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/5.jpg)
Staining the Gel(destain in water)
RNA band (18 bases)
DNA band 35 bases
![Page 6: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/6.jpg)
Use of Fiber Optic Technology
![Page 7: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/7.jpg)
Detector
Halogen Source
Use of Fiber Optic Technology
(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/Instrumentation/OceanOptics/OOIntro.htm
![Page 8: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/8.jpg)
Dyes
S
N
NH3C
CH3Cl
N
CH3
CH3S
N CH3
NH2NH3C
CH3Cl
Cresyl Violet Toluidine Blue OMethylene Blue
O
N
NH2H2NCH3COO
Azure A Azure B Azure C Thionin
Nile Blue A Brilliant Cresyl BlueCoomassie Brilliant Blue
![Page 9: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/9.jpg)
General Testing of Dyes
Strips of Gel Stained with Methylene Blue, Azure A, and Azure C prior to destaining
RNA
DNA
![Page 10: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/10.jpg)
Azure B
––– RNA
––– DNA
![Page 11: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/11.jpg)
Azure A
––– RNA
––– DNA
![Page 12: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/12.jpg)
Azure C
––– RNA
––– DNA
![Page 13: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/13.jpg)
Toluidine Blue O
––– RNA
––– DNA
S
N
NH3C
CH3Cl
N
CH3
CH3
![Page 14: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/14.jpg)
Cresyl Violet
––– RNA
––– DNA
O
N
NH2H2NCH3COO
![Page 15: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/15.jpg)
Brilliant Cresyl Blue
––– RNA
––– DNA
![Page 16: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/16.jpg)
Methylene Blue
––– RNA
––– DNA
S
N CH3
NH2NH3C
CH3Cl
![Page 17: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/17.jpg)
Thionin
––– RNA
––– DNA
![Page 18: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/18.jpg)
Coomassie and Nile Blue A
Coomassie Nile Blue A
![Page 19: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/19.jpg)
Dyes Chosen for Quantitative Analysis
S
N
NH3C
CH3Cl
N
CH3
CH3S
N CH3
NH2NH3C
CH3Cl
Cresyl Violet Toluidine Blue OMethylene Blue
O
N
NH2H2NCH3COO
Azure A Azure B Azure C Thionin
Nile Blue A Brilliant Cresyl BlueCoomassie Brilliant Blue
![Page 20: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/20.jpg)
Thionin
DNA
RNA
10 – 200 picomoles
![Page 21: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/21.jpg)
Azure B
RNA
DNA
10 – 200 picomoles
![Page 22: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/22.jpg)
Cresyl Violet
RNA
DNA
10 – 200 picomoles
![Page 23: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/23.jpg)
Methylene Blue
DNA
RNA
DNARNA
10 – 200 picomoles
![Page 24: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/24.jpg)
Quantitative Data Azure B DNAAbs orbanc e vs . Amount of DNA for Azure B at 601.43 nm
y = 0.0013x + 0.1825R ² = 0.866
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 50 100 150 200 250Amount (pmol)
Abso
rbanc
e
![Page 25: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/25.jpg)
Quantitative Data Methylene Blue DNAAbs orbanc e vs . Amount of DNA for Methylene B lue at 670.54 nm
y = 0.0006x + 0.3429R ² = 0.6061
y = 0.0006x + 0.24R ² = 0.3414
0
0.1
0.2
0.3
0.4
0.5
0.6
0 50 100 150 200 250 300
Amount (pmol)
Abso
rban
ce
Trial 1
Trial 2
![Page 26: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/26.jpg)
Quantitative Data Cresyl Violet DNAAbs orbanc e vs . Amount of DNA for C res yl Violet at 560 nm
y = 0.0003x + 0.1338R ² = 0.3152
y = 0.0005x + 0.145R ² = 0.861
0
0.05
0.1
0.15
0.2
0.25
0.3
0 50 100 150 200 250 300Amount (pmol)
Abso
rban
ce
Trial 1
Trial 2
![Page 27: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/27.jpg)
Quantitative Data Thionin DNAAbs orbanc e vs . Amount of DNA for T hionin at 560 nm
y = 0.0009x + 0.1818R ² = 0.8071
y = 0.0008x + 0.2441R ² = 0.6753
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 50 100 150 200 250 300
Amount (pmol)
Abso
rban
ce
Trial 1
Trial 2
![Page 28: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/28.jpg)
Corrected Thionin DNA DataAbs orbanc e vs . Amount of DNA for T hionin at 560 nm (C orrec ted)
y = 0.0005x + 0.2647R ² = 0.9979
y = 3E -05x + 0.3645R ² = 0.75
y = 0.003x + 0.0703R ² = 0.9832
y = 0.0022x + 0.1775R ² = 0.9906
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 50 100 150 200 250 300
Amount (pmol)
Abso
rban
ce
![Page 29: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/29.jpg)
Quantitative Data Azure B RNAAbs orbanc e vs . Amount of R NA for Azure B at 600.85 nm
y = 0.0009x + 0.2181R ² = 0.6103
y = 0.002x + 0.1082R ² = 0.9876
0
0.1
0.2
0.3
0.4
0.5
0.6
0 50 100 150 200 250 300
Amount (pmol)
Abso
rban
ce
Trial 1
Trial 2
![Page 30: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/30.jpg)
Corrected Azure B RNA DataAbs orbanc e vs . Amount of R NA for Azure B at 600.85 nm
(C orrec ted)
y = 0.0029x + 0.0925R ² = 0.9826
y = 0.002x + 0.1082R 2 = 0.9876
0
0.1
0.2
0.3
0.4
0.5
0.6
0 50 100 150 200 250
Amount (pmol)
Abso
rban
ce
Trial 1 (corrected)
Trial 2
![Page 31: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/31.jpg)
Quantitative Data Cresyl Violet RNA
Abs orbanc e vs . Amount of R NA for C res yl Violet at 560 nm
y = 0.0008x + 0.1219R ² = 0.9552
y = 0.0011x + 0.0957R ² = 0.8671
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 50 100 150 200 250 300
Amount (pmol)
Abso
rban
ce
Trial 1
Trial 2
![Page 32: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/32.jpg)
Quantitative Data Thionin RNAAbs orbanc e vs . Amount of R NA for T hionin at 560.04 nm
y = 0.0002x + 0.1259R ² = 0.1756
y = 0.0017x + 0.1176R ² = 0.8422
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 50 100 150 200 250 300
Amount (pmol)
Abso
rban
ce
Trial 1
Trial 2
![Page 33: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/33.jpg)
Quantitative Data Methylene Blue RNAAbs orbanc e vs . Amount of R NA for Methylene B lue
y = 0.0017x + 0.1162R ² = 0.9374
y = 0.0009x + 0.1939R ² = 0.9054
y = 0.0014x + 0.165R ² = 0.8657
y = 0.001x + 0.1485R 2 = 0.9638
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 50 100 150 200 250 300
Amount (pmol)
Abso
rban
ce
672 nm- Trial 2
601 nm- Trial 1
601 nm- Trial 2
672 nm-Trial 1
![Page 34: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/34.jpg)
Corrected Methylene Blue RNA DataAbs orbanc e vs . Amount of R NA for Methylene B lue (C orrec ted)
y = 0.0017x + 0.144R ² = 0.9894y = 0.0027x + 0.1136
R ² = 0.9335
y = 0.0016x + 0.1145R 2 = 0.9874
y = 0.0022x + 0.0966R 2 = 0.9569
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 20 40 60 80 100 120
Amount (pmol)
Abso
rban
ce
601 nm- Trial 1
601 nm- Trial 2
672 nm- Trial 1
672 nm- Trial 2
![Page 35: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/35.jpg)
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
![Page 36: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/36.jpg)
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
![Page 37: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/37.jpg)
AcknowledgementsDr. MiyamotoDr. Paul Quinn
CounselorsSchering-Plough Foundation
NovartisThe Dorr Foundation
The Jennifer A. Chalsty FoundationThe Edward W. and Stella C. Van Houten Memorial Fund
The Jewish Communal Fund Laura and John Overdeck
NJGSS Alumni and Parents 1984-2008
![Page 38: Getting the Stain IN A Quantitative Analysis of DNA and RNA Staining with Various Dyes Dr. Amber Charlebois Teaching Assistant: Jeremy Tang Rupak Bhuyan](https://reader036.vdocument.in/reader036/viewer/2022062500/5697bfe11a28abf838cb39e9/html5/thumbnails/38.jpg)
Questions?