getting the stain in a quantitative analysis of dna and rna staining with various dyes dr. amber...
Post on 29-Jan-2016
215 Views
Preview:
TRANSCRIPT
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
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
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
Loading Dye RNA DNA
Loading Dye
Loading Dye RNA
RNADNA
Loading Dye
RNA DNA
(Begin Run)
Staining the Gel(destain in water)
RNA band (18 bases)
DNA band 35 bases
Use of Fiber Optic Technology
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
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
General Testing of Dyes
Strips of Gel Stained with Methylene Blue, Azure A, and Azure C prior to destaining
RNA
DNA
Azure B
––– RNA
––– DNA
Azure A
––– RNA
––– DNA
Azure C
––– RNA
––– DNA
Toluidine Blue O
––– RNA
––– DNA
S
N
NH3C
CH3Cl
N
CH3
CH3
Cresyl Violet
––– RNA
––– DNA
O
N
NH2H2NCH3COO
Brilliant Cresyl Blue
––– RNA
––– DNA
Methylene Blue
––– RNA
––– DNA
S
N CH3
NH2NH3C
CH3Cl
Thionin
––– RNA
––– DNA
Coomassie and Nile Blue A
Coomassie Nile Blue A
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
Thionin
DNA
RNA
10 – 200 picomoles
Azure B
RNA
DNA
10 – 200 picomoles
Cresyl Violet
RNA
DNA
10 – 200 picomoles
Methylene Blue
DNA
RNA
DNARNA
10 – 200 picomoles
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Questions?
top related