Genomics & Proteomics - Biotium

Copy and paste this link to your website, so they can see this document directly without any plugins.



Keywords

with, GelRed™, GelGreen™, dsDNA, SYBR®, EvaGreen®, PAGE, reactions, qPCR, AccuBlue™, Quantitation, Green, from, Figure, Master, High, dyes, fluorescence, than, that, cell, detection, AccuLite™, Sensitivity, using, compatible, www.biotium.com, Molecular, other, more

Transcript

PCR
• qPCR Master Mixes with EvaGreen® dye ... pp. 2-3
• Cheetah™ hot-start Taq ... p. 3
• HotStart & WarmStart™ Polymerase Modification Kits ... p. 4
• EvaEZ™ DNA Polymerase Activity Assay ... p. 4
DNA and protein quantitation
• AccuBlue™ and AccuClear™ DNA Quantitation Assays ... pp. 6-7
• AccuOrange™ Protein Quantitation Assay ... p. 8
• AccuLite™ handheld mini-fluorometers ... p. 9
Gel stains
• GelRed™ & GelGreen™ safe nucleic acid stains ... pp. 10-11
• PAGE GelRed™ & PAGE GelGreen™ ... p. 12
• Lumitein™ Protein Gel Stain ... p. 13
Microbe Detection by PCR
• PMA™ and PMAxx™ for bacterial viability PCR ... pp. 14-15
• PMA-Lite™ Photolysis Device ... p. 15
Products for
Molecular
Biology
2 Molecular Biology Products • www.biotium.com
EvaGreen® Dye for qPCR
Sensitive, safe, and stable
FEATURES OF EVAGREEN® DYE
• Safer and more environmentally friendly
• High sensitivity
• Detected using SYBR® Green instrument settings
• Direct visualization of PCR product in gels
• Extremely thermostable (see reference: Mil. Med. 2014
179(6):626-32).
• The only qPCR dye used in droplet digital PCR
(ddPCR)
EvaGreen® dye is a next-generation DNA-binding dye with features ideal for use in quantitative real-time PCR (qPCR) and other applications. Biotium scientists designed the dye with
several essential dye properties in mind, including PCR inhibition, dye
safety, stability, and fluorescence spectra of the dye. The result of that
effort is a dye superior to other dyes such as SYBR® Green I for PCR
and melt curve analysis.
Superior PCR performance
EvaGreen® dye binds to dsDNA via a novel “release-ondemand” mechanism, which permits the use of a relatively high dye
concentration without inhibiting PCR (Figure 1). The dye is constructed
of two monomeric DNA-binding dyes linked by a flexible spacer. The
dimeric dye randomly shifts between an inactive looped conformation
that does not bind DNA at low DNA concentrations at the beginning of
a PCR reaction, and an active conformation that becomes stabilized as
DNA concentration increases during PCR. Consequently, EvaGreen®
can be used at relatively high dye concentrations, resulting in improved
sensitivity and more accurate melt curve analysis compared to
other qPCR dyes such as SYBR® Green. EvaGreen® dye is also
compatible with microfluidics-based qPCR platforms and isothermal
EvaGreen® is a registered trademark of Biotium, Inc. EvaGreen dye technologies are covered by granted US and/or international patents. Amplitaq Gold® and TaqMan® are registered trademarks of Roche Molecular Systems, Inc. SYBR® is a
registered trademark of Life Technologies. Practicing HRM may require a license from Idaho Technologies, Inc.
Figure 1. EvaGreen® dye binds to dsDNA via a “release-on-demand” mechanism.
DNA amplification methods. EvaGreen® is detected using the same
instrument settings as SYBR® Green I.
An added benefit of EvaGreen® dye is that EvaGreen® dye
in the PCR reaction doubles as a DNA prestain, permitting direct
visualization of DNA bands following gel electrophoresis without any
further gel staining.
Safer handling and disposal
A unique feature of EvaGreen® dye is its safety. Handling and
disposal of qPCR master mixes may pose health and environmental
risks. For example, SYBR® Green I, a popular PCR dye, has been
found to be even more environmentally toxic than ethidium bromide, a
well-known mutagen (Ohta et al., 2001). With this in mind, Biotium’s
scientists designed EvaGreen® dye such that it cannot cross cell
membranes, thus preventing the dye from binding genomic DNA
in living cells (Figure 2). Independent labs have confirmed that
EvaGreen® dye is nonmutagenic, noncytotoxic and safe to aquatic
life, and classified as non-hazardous waste. A detailed safety report
for EvaGreen® is available for download from www.biotium.com.
EvaGreen® has been cited in more than 2500 publications to date
(Google Scholar search).
Cell Membrane Impermeability Means a Safer Dye
Figure 2. Comparison of cell membrane permeability between EvaGreen®
dye and SYBR® Green I. HeLa cells were incubated with SYBR® Green I
(1.2 µM) or EvaGreen® dye (1.2 µM) at 37 oC. Images were taken following incubation for 5 and 30 minutes. SYBR® Green I entered cells rapidly
while EvaGreen® dye appeared membrane-impermeable as evident from
the absence of cell nuclear staining. Image taken with long photo-exposure
time revealed that EvaGreen® dye only associated with cell membranes.
Molecular Biology Products • www.biotium.com 3
Figure 4. Comparison among Fast-Plus EvaGreen® qPCR Master Mix from Biotium
and two fast SYBR® Green master mixes from two leading companies (company A
and company Q) under similar condition. The inset is an enlarged view of the area
near the baseline for better viewing the curve patterns of the weaker signals of the
two SYBR-based master mixes. Amplicon: ATPG fragment of human genomic DNA;
instrument: ABI 7900 Fast.
CheetahTM Taq & EvaGreen® qPCR Master Mixes
Fast EvaGreen® qPCR Master Mixes
NEW! Forget-Me-NotTM qPCR Master Mixes
Figure 6. PCR tubes containing Forget-Me-Not™ qPCR Master Mix (1X) on
left and Forget-Me-Not qPCR Master Mix (1X) plus DNA template in Template
Buffer on the right.
Two Color Tracking to Minimize Errors
Convenient, ready-to-use qPCR Master Mixes feature bright and
safe EvaGreen® dye and our proprietary CheetahTM Taq hot-start
polymerase.
We also offer Fast Probe Master Mixes featuring CheetahTM Taq
without dye, for probe-based PCR reactions. Our Fast Probe
Master Mixes are used extensively in microfluidics-based PCR
platforms.
Figure 5. Real-time PCR data comparing Forget-Me-Not™ (blue line) with Biotium’s Fast EvaGreen® (green line) and
Qiagen's QuantiNova® SYBR® Green (black line) master mixes. Amplification curves on linear scale. EvaGreen® dyebased master mixes yield higher signal compared to the SYBR® Green-based mix.
Figure 7. Melt curve analysis comparing Forget-Me-Not™ (blue line) with Quanta's PerfeCTa SYBR® Green
FastMix (green line).
0
1
2
3
4
15 25 35 45
A
FU
Cycle Number
Fast EvaGreen
Company A
Company Q
0
1
15 25 35 45
A
FU
Cycle Number
Forget-Me-Not™
Fast EvaGreen®
QuantiNova SYBR® Green
Eliminate costly pipeting errors!
Biotium’s new Forget-Me-NotTM qPCR Master Mixes
feature the same EvaGreen® dye and CheetahTM Taq
as our Fast EvaGreen Master Mixes, with the added
benefit of two-color tracking:
• Light blue qPCR Master Mix
• Dark blue after addition of template buffer
Forget-Me-Not™
PerfeCTa SYBR® Green FastMix
Superior Melt Curve Analysis
Brighter signal than SYBR® Green
Outstanding qPCR performance
0
20
40
60
80
100
0 1 2 5 10 20
R
el at iv e A
ct iv ity
(%
)
Minutes at 95°C
Cheetah Taq
AmpliTaq Gold
Figure 3. Cheetah™ Taq requires only 2 minutes
of hot-start for full recovery of activity. Comparison
of hot-start recovery of polymerase activity for
Cheetah™ Taq and AmpliTaq Gold® following
incubation at 95°C in 50 mM pH 8.0 Tris.
CheetahTM Taq is Biotium’s proprietary chemically-modified hot-start DNA polymerase. Unlike AmpliTaq Gold®, which takes 10 minutes or longer to activate,
CheetahTM Taq is fully recovered in 2 minutes with high activity,
making it particularly suitable for fast PCR.
Cheetah™ Taq is covered by a granted US patent.
4 Molecular Biology Products • www.biotium.com
EvaEZ™ Fluorometric Polymerase Activity Assay
• Non-radioactive assay for DNA acid polymerases
• Uses safe and sensitive EvaGreen™ dye
• Assay enzyme activity between 4oC-75oC.
• Confirm hot-start or warm-start modification of DNA
polymerases (Figure 8a)
Determine polymerase activity of:
• Taq, Pfu, Vent®, Phusion®
• AMV
• Bst
• Phi29
• MMLV, SuperScript®
• T4 & T7 DNA polymerases
• E. coli DNA polymerase I, Klenow fragment
HotStart Polymerase Modification Kit provides an easy way to reversibly modify lysine residues of thermostable DNA polymerases, rendering the enzyme inactive. The modification is reversed after heating to >90oC. Hot-start modification of DNA polymerase for PCR prevents amplification of non-specific PCR products due to low stringency annealing of primers at low temperature during reaction assembly.
The WarmStart™ Enzyme Modification Kit allows temperature control of enzyme activity to be applied to non-thermostable enzymes like reverse
transcriptase. Like HotStart, the enzyme is inactivated by modification of lysine residues with a chemical modifier. Protein activity is restored by
heating to 45oC or higher.
Phusion® is a registered trademark of Thermo Fisher Scientific. SuperScript® is a registered trademark of Life Technologies. Vent® is a registered trademark of New England BioLabs. HotStart and WarmStart modification kits are covered by pending
U.S. and international patents.
Time (min)
Fl uo re sc en ce b c a Figure 8. A. EvaEZ™ Polymerase Activity assay showing Taq polymerase activity
without modification (a), after modification with the HotStart Polymerase Modification
Kit (b), and after reactivation of the modified enzyme by heating to 90oC (c). B.
Lumitein-stained non-denaturing acrylamide gel showing increased electrophoretic
mobility of Taq polymerase after modification (lane 2) compared to unmodified
enzyme (lane 1).
WarmStart™ Enzyme Modification Kit
• Novel chemical modifier for non-thermostable enzymes
• Turn off enzyme activity at room temperature
• Regain activity by heating to
45-60oC
Applications:
• Reverse transcriptase
• Bst DNA polymerase
• E. coli DNA polymerase I
• Restriction enzymes
• Nucleases
• Proteases
HotStart Polymerase Modification Kit
• Hot-start any thermostable DNA
polymerase with the same patented
technology used in Cheetah™ Taq
• Prevent primer-dimer formation and mis-priming
• Fast hot-start activation, 2 minutes at 95oC
• Includes Lumitein™ protein gel stain (p. 13) to confirm
modification by PAGE gel electrophoresis (Figure 8b)
A
Modified
Unmodified
B 1 2
DNA Polymerase Modification and Activity Kits
Molecular Biology Products • www.biotium.com 5
PCR products ordering information
Description Size Cat. No.
Forget-Me-Not™ qPCR Master Mix
Trial size, 100 reactions 31041-T
500 reactions 31041-1
Forget-Me-Not™ qPCR Master Mix with ROX
Trial size, 100 reactions 31042-T
500 reactions 31042-1
Fast EvaGreen® qPCR Master Mix
Trial size, 100 reactions 31003-T
200 reactions 31003
500 reactions 31003-1
5000 reactions 31003-2
Fast Plus EvaGreen® qPCR Master Mix
Trial size, 100 reactions 31020-T
200 reactions 31020
500 reactions 31020-1
5000 reactions 31020-2
Fast Plus EvaGreen® qPCR Master Mix, Low
ROX
Trial size, 100 reactions 31014-T
200 reactions 31014
500 reactions 31014-1
5000 reactions 31014-2
Fast Plus EvaGreen® qPCR Master Mix, High
ROX
Trial size, 100 reactions 31015-T
200 reactions 31015
500 reactions 31015-1
5000 reactions 31015-2
Fast Probe Master Mix
Trial size, 100 reactions 31005-T
200 reactions 31005
500 reactions 31005-1
5000 reactions 31005-2
Fast Probe Master Mix, High ROX Trial size, 100 reactions 31016-T
200 reactions 31016
500 reactions 31016-1
5000 reactions 31016-2
qPCR Master Mixes
Description Size Cat. No.
EvaGreen® Dye, 20X in Water
Trial size, 1 mL 31000-T
5 x 1 mL 31000
EvaGreen® Dye, 2000X in DMSO 50 uL 31019
dNTP mix, 10 mM each 5 x 1 mL 40054
dNTP set, 100 mM each Set of 1 mL each 40052
Cheetah™ Taq 500 U 29050
ROX Passive Reference Dye, 25 uM in TE 5 x 1 mL 29052
PCR Reagents sold separately
Description Size Cat. No.
HotStart Polymerase Modification Kit
Trial size, sufficient to modify
0.1 mg polymerase
29054-T
Sufficient to modify 0.5 mg
polymerase
29054
WarmStart™ Enzyme Modification Kit
Trial size, sufficient to modify
0.1 mg polymerase
29053-T
Sufficient to modify 0.5 mg
polymerase
29053
EvaEZ™ Fluorometric Polymerase Activity
Assay Kit
200 reactions 29051
Enzyme modification and assay kits
6 Molecular Biology Products • www.biotium.com
AccuBlue™ and AccuClear™ dsDNA quantitation assays allow precise quantitation of purified dsDNA samples across a wide range of concentrations. Unlike absorbance-based nucleic acid quantitation, fluorescent DNA binding dyes are highly sensitive and selective for double-stranded DNA and provide a more accurate DNA concentration in the presence of contaminating RNA and other common
contaminants including free nucleotides, protein, detergents and salts. Biotium offers four dsDNA quantitation kits for different sample
concentration ranges.
All kits are compatible with Biotium’s compact and affordable AccuLite™ Mini Fluorometers (see p. 9).
Advantages of AccuBlueTM and AccuClearTM :
• Wide dynamic range
• High precision ideal for use in NGS
applications
• Best sensitivity on the market
• Safer than PicoGreen® (AccuBlueTM HS*)
AccuBlue™ Broad Range
• Linear range: 2-2000 ng dsDNA
• Blue fluorescence (Ex/Em: 350/460 nm)
• Assay can be extended to 4000 ng dsDNA with
minor loss of linearity
• Blue fluorescence detection, compatible with
fluorescence microplate reader and Biotium’s
AccuLite™ 350 handheld fluorometer
AccuBlue™ High Sensitivity
• Linear range: 0.2-100 ng dsDNA
• Green fluorescence (Ex/Em: 485/530 nm)
• Membrane-impermeable dye is non-toxic and
non-mutagenic, for safer handling and easy
disposal
• Green fluorescence detection compatible with
fluorescence microplate reader, NanoDrop®
fluorospectrometer, Biotium’s AccuLite™ 470
handheld fluorometer and other handheld
fluorometers like Qubit®, QuantusTM and
QuantiFluor-P™
AccuClear™ Ultra High Sensitivity
• Linear range: 0.03-250 ng dsDNA
• Green fluorescence (Ex/Em: 468/507 nm)
• First-class sensitivity and dynamic range
• Novel green fluorescent dye is a perfect match for
blue LED excitation sources
• Green fluorescence detection compatible with
fluorescence microplate readers, Biotium’s
AccuLite™ 470 handheld fluorometer, or
NanoDrop® fluorospectrometer
AccuBlueTM and AccuClearTM dsDNA Quantitation kits
NEW! AccuBlue™ NextGen
• Linear range: 2.5 pg-3 ng dsDNA
• Green fluorescence (Ex/Em: 468/507 nm)
• The most sensitive and accurate dsDNA
quantitation reagent on the market
• Ideal for quantification of low-concentration or
precious samples
• Fluorescence detection compatible with
fluorescence microplate readers, Biotium’s
AccuLite™ 470 handheld fluorometer, or
NanoDrop® fluorospectrometer
Figure 9. Chart comparing the linear detection ranges for each dsDNA Quantitation Kit. The units are ng of
dsDNA per 200 uL reaction. Shown in log scale.
*AccuBlue™ HS has been demonstrated by independent labs to be safer than PicoGreen®. Lower toxicity is due to reduced membrane permeability.
AccuBlue™ is covered by granted US and/or international patents. AccuClear™ is covered by a pending patent. AccuBlue™, AccuClear™, and AccuLite™ are trademarks of Biotium, Inc.; PicoGreen®, Qubit® and Quant-iT® are trademarks of Life
technologies; NanoDrop® is a registered trademark of Thermo Scientific.
Linear detection ranges of dsDNA Quantitation Assays
Molecular Biology Products • www.biotium.com 7
AccuBlue™ High Sensitivity (0.2-100 ng)
R² = 0.9994
0
500
1000
1500
2000
2500
0 20 40 60 80 100
Fl uo re sc en ce DNA (ng/well)
0
25
50
75
100
0 1 2 3 4 5
Fl uo re sc en ce DNA (ng/well)
AccuClear™ Ultra High Sensitivity (.03-250 ng)
R² = 0.998
0
2000
4000
6000
8000
10000
12000
14000
0 50 100 150 200 250
Fl uo re se nc e DNA ng/well
0
20
40
60
0 0.2 0.4 0.6 0.8 1
Fl uo re se nc e DNA ng/well
Figure 13: Two-fold dilutions of calf thymus DNA were assayed using AccuBlue™ or QuantiT™ Broad Range assay kits. AccuBlue™ has a wider dynamic range than the Quant-iT™.
AccuBlue™ Broad Range vs. Quant-iT™ Broad Range (2-4000 ng)
0
3000
6000
9000
0 1000 2000 3000 4000
Fl uo re sc en ce DNA (ng/well)
AccuBlue™
Quant-iT™
Figure 12: Standard curve of calf thymus DNA assayed using the AccuBlue™ High
Sensitivity Kit and read on a microplate reader (Ex/Em 485/530). Inset shows the
lower end of the titration.
The most sensitive and accurate dsDNA quantitation reagents on the market!
Accurate and reproducible
results every time
AccuBlueTM and AccuClearTM dsDNA Quantitation kits
AccuBlue™ NextGen (2.5-3000 pg)
Figure 11: Standard curve of calf thymus DNA assayed using the AccuClear™ Ultra
High Sensitivity Kit and read on a microplate reader (Ex/Em 460/507). Inset shows the
lower end of the titration.
Figure 10: Standard curve of calf thymus DNA assayed using the AccuBlue™ NextGen
Assay Kit and read on a microplate reader (Ex/Em 468/507). Inset shows the lower
end of the titration, with accuracy down to 2.5 pg of DNA, depending on the instrument.
Linear Range Description Cat. No. Unit Size
Most Sensitive
2.5-3000 pg
AccuBlue™ NextGen dsDNA Quantitation Kit with 1 DNA Standard 31060, 31060-T 1000, 200 assays
Ultra High Sensitivity
.03-250 ng
AccuClear™ Ultra High Sensitivity dsDNA Quantitation Kit with 7 DNA Standards 31028 1000 Assays
AccuClear™ Ultra High Sensitivity dsDNA Quantitation Kit with 1 DNA Standard 31029 4000 Assays
AccuClear™ Ultra High Sensitivity dsDNA Quantitation Solution 31027, 31027-T 1000, 200 assays
High Sensitivity
0.2-100 ng
AccuBlue™ High Sensitivity dsDNA Quantitation Kit with DNA Standard, Trial Size 31006-T 200 Assays
AccuBlue™ High Sensitivity dsDNA Quantitation Kit with 8 DNA Standards 31006 1000 Assays
AccuBlue™ High Sensitivity dsDNA Quantitation Solution 31008, 31008-T 1000, 200 assays
Broad Range
2-2000 ng
AccuBlue™ Broad Range dsDNA Quantitation Kit with DNA Standard, Trial Size 31007-T 200 Assays
AccuBlue™ Broad Range dsDNA Quantitation Kit with 9 DNA Standards 31007 1000 Assays
AccuBlue™ Broad Range dsDNA Quantitation Solution 31009, 31009-T 1000, 200 assays
Stand-alone dsDNA
standard sets
AccuBlue™ Broad Range dsDNA Standards, Set of Nine (0-200 ng/uL calf thymus dsDNA) 31007C 0.5 mL each
AccuBlue™ High Sensitivity dsDNA Standards, Set of Eight (0-10 ng/uL calf thymus dsDNA) 31006C 0.5 mL each
Ordering Information
Fl uo re sc en ce 8 Molecular Biology Products • www.biotium.com
AccuOrangeTM Protein Quantitation Kit
AccuOrange™ Protein Quantitation Kit is a highly sensitive fluorescence-based assay for quantitating purified protein samples in 96-well format. The detection range of the assay is 0.1-15 ug/mL protein. AccuOrange is much more sensitive than traditional protein
quantitation assays such as BCA, Bradford and Lowry, and shows superior linearity and reproducibility compared to the NanoOrange®
protein quantitation assay (Figure 14). The assay shows minimal variability between different proteins, and has stable fluorescence signal
for up to 16 hours.
AccuOrange™ is recommended for quantitating purified protein or antibody samples. The AccuOrange™ assay has low tolerance for
non-ionic detergents, and is not recommended for use with cell lysates containing Triton X-100, sodium deoxycholate, CHAPs, or other
non-ionic detergents. The assay can tolerate up to 0.01% SDS (final concentration in assay).
FEATURES
• Ex/Em: 480/598 nm
• Linear detection range: 0.1-15 ug/mL protein
• 200 uL microplate assay
• Minimal protein-protein variation
• Fluorescence signal stable for at least 16 hours
• Compatible with reducing agents, amino acids, nucleic acids,
and imidazole
• For use with purified protein or antibody samples
• Compatible with AccuLite™ 470 Mini-Fluorometer (p. 9)
Description Cat. No. Size
AccuOrange™ Protein Quantitation Kit
30071-T 200 assays
30071 2000 assays
Ordering Information
AccuOrange
R² = 0.9981
NanoOrange
R² = 0.9453
0
500
1000
1500
2000
2500
0 5 10 15
Fl uo re sc en ce BSA (ug/mL)
AccuOrange
NanoOrange
Figure 14. AccuOrange™ shows better linearity and reproducibility compared to
NanoOrange® Protein Quantitation assay. BSA titration was performed in triplicate
using AccuOrange™ Protein Quantitation Kit or NanoOrange® Protein Quantitation
Kit from Life Technologies according to manufacturer’s protocol and read on a
microplate reader at the recommended wavelengths for each assay. Inset shows
the lower end of the curve. Error bars represent standard deviation of the mean for
triplicate samples.
0
200
400
0 0.5 1 1.5
Fl uo re sc en ce BSA (ug/mL)
Assay Type Detection Range (microplate assay) Comments
AccuOrange™ 0.1-15 ug/mL
• Fluorescence detection (480/598 nm)
• Highly linear
• Signal stable for at least 16 hours
• Compatible with reducing agents
• Not compatible with detergents
NanoOrange® 0.1-10 ug/mL
• Fluorescence detection (470/570 nm)
• Non-linear
• Fluorescence stable for 6 hours
• Compatible with reducing agents
• Not compatible with detergents
Modified Lowry 1-1500 ug/mL
• Absorbance detection (750 nm)
• Non-linear
• Not compatible with reducing agents
• Not compatible with detergents
BCA 20-2000 ug/mL
• Absorbance detection (562 nm)
• Highly linear
• Signal not stable over time
• Not compatible with reducing agents
• Compatible with detergents
Bradford
(Coomassie) 50-500 ug/mL
• Absorbance detection (595 nm)
• Signal not stable over time
• Non-linear
• Compatible with reducing agents
• Not compatible with detergents
Pierce® 660 nm 50-2000 ug/mL
• Absorbance detection (660 nm)
• Non-linear
• Compatible with reducing agents
• Compatible with detergents
A280 50-2000 ug/mL
• Absorbance detection (280 nm)
• High protein-protein variability
• Contaminants such as nucleic acids
can affect results
Table 1. Comparison of AccuOrange™ with other protein quantitation assays
Highly sensitive, fluorometric protein assay
NanoOrange® is a registered trademark of Life Technologies.
Molecular Biology Products • www.biotium.com 9
AccuLite™ Mini Fluorometers
• Accept 200 uL PCR tubes
• LCD touch-screen display
• USB interface for data management
• Compact (185 x 90 x 35 mm, 10 oz)
• Greater than 6 logs of dynamic range
• Include programs for use with AccuBlue™ and
AccuClear™ dsDNA quantitation assays
• Power Supply: 4 AA batteries or a 5V DC adapter
• AccuLite™ 350: 365-370 nm LED excitation
460+/-20 nm emission
• AccuLite™ 470: 465-475 nm LED excitation
540+/-30 nm emission
Description Cat. No. Unit Size
AccuLite™ Mini
Fluorometers
AccuLite™ 350 Mini Fluorometer E90000 Each
AccuLite™ 470 Mini Fluorometer E90001 Each
Ordering Information
AccuLiteTM Handheld Fluorometers
Assays for AccuLite™ 350:
AccuBlue™ Broad Range dsDNA Quantitation Assay
• Linear range: 2-2000 ng dsDNA
• Assay can be extended to 4000 ng dsDNA with minor loss
of linearity
Assays for AccuLite™ 470:
AccuBlue™ NextGen dsDNA Quantitation Assay
• Linear range: 2.5-3000 pg dsDNA
• Green fluorescence (Ex/Em: 468/507 nm)
• Unrivaled sensitivity
AccuClear™ Ultra High Sensitivity dsDNA Quantitation Assay
• Linear range: 0.03-250 ng dsDNA
• Green fluorescence (Ex/Em: 468/507 nm)
• Combined sensitivity and dynamic range
AccuBlue™ High Sensitivity dsDNA Quantitation Assay
• Linear range: 0.2-100 ng dsDNA
• Membrane-impermeable dye is non-toxic and nonmutagenic, for safer handling and easy disposal
AccuOrange™ Protein Quantitation Kit
• Linear range: 0.1-15 ug/mL protein
• For quantitation of purified protein or antibody samples
• Tolerates up to 0.1% SDS (final concentration in assay)
AccuLite™ Mini Fluorometers are portable instruments designed for multipurpose fluorescence measurements. The instruments are simple to use, lightweight, and can be powered by either DC power adapter or battery, making them an excellent choice for field
studies and laboratory measurements.
10 Molecular Biology Products • www.biotium.com
GelRed™ and GelGreen™ are next-generation fluorescent nucleic acid gel stains designed to replace the highly toxic
ethidium bromide (EtBr). Developed by scientists at Biotium,
GelRed™ and GelGreen™ are superior to EtBr and other EtBr
alternatives by having a combination of low toxicity, high sensitivity
and exceptional stability.
EtBr has been the predominant dye used for nucleic acid
gel staining for decades because of its low price and generally
sufficient sensitivity. However, EtBr is a highly mutagenic chemical.
The safety hazard and costs associated with decontamination
and waste disposal can ultimately make the dye expensive and
inconvenient to use. For this reason, alternative gel stains, such as
SYBR® dyes, have become commercially available in recent years.
While these alternative dyes have reduced mutagenicity, they
sacrifice sensitivity and stability. For example, SYBR® Safe has
very limited sensitivity while SYBR® Green and SYBR® Gold are
much less stable than EtBr. SYBR® dyes also enter cells rapidly
to stain mitochondria and nuclear DNA, making it more likely for
the dyes to be harmful to cells. Indeed, SYBR® Green I has been
shown to strongly potentiate DNA mutation caused by UV light and
other mutagens (Ohta, et al. 2001).
Safer options for gel staining
To make safer gel stains, scientists at Biotium used a novel
yet very simple concept: reducing genotoxicity by preventing the
dyes from entering living cells. We believe that the mutagenicity
of a DNA-binding dye can be greatly reduced by denying it
access to genomic DNA in living cells. Thus, we engineered the
chemical structures of GelRed™ and GelGreen™ such that the
dyes are incapable of crossing cell membranes. Ames tests have
Figure 15. GelRed™ and GelGreen™ are more sensitive than EtBr and SYBR® Safe.
Left: Comparison of GelRed™ and ethidium bromide (EtBr) in precast gel staining using
1% agarose gel in TBE buffer. Right: Comparison of GelGreen™ and SYBR® Safe in
post gel staining using 1% agarose gel in TBE buffer. Two-fold serial dilutions of 1 kb
Plus DNA Ladder from Invitrogen were loaded onto each gel in 4 lanes in the amounts
of 200 ng, 100 ng, 50 ng and 25 ng, respectively, from left to right.
GelRed™EtBr GelGreen™SYBR® Safe
confirmed that GelRed™ and GelGreen™ are nonmutagenic at
concentrations well above the concentrations used for gel staining.
Furthermore, environmental safety tests showed that GelRed™
and GelGreen™ are non-toxic to aquatic life. Because of this,
GelRed™ and GelGreen™ are classified as non-hazardous waste,
and can be disposed as regular trash or down the drain. For more
information, please download the GelRed™/GelGreen™ Safety
Report at www.biotium.com.
Superior sensitivity
GelRed™ and GelGreen™ are highly sensitive for precast
or post-electrophoresis gel staining. Designed primarily for use
with a 312/302 nm UV transilluminator, GelRed™ is much more
sensitive than EtBr. GelGreen™ is compatible with visible blue
light transilluminators and UV to blue light converter plates,
which protect users and DNA samples from UV light exposure.
Using blue light excitation instead of UV when excising DNA from
preparative gels greatly improves downstream cloning efficiency
(Gründemann and Schömig 1996). GelGreen™ is spectrally
similar to SYBR® Safe, but is far more sensitive than the latter.
Another advantage of GelRed™ and GelGreen™ is their
chemical stability. You can handle the two dyes the same way
as EtBr. This means that the dyes are perfectly stable in water at
room temperature for long-term storage, and they can be added to
molten agarose for making precast gels. Both dyes are also very
photostable and can be handled under normal room light.
Biotium gel extraction kits and DNA ladders are optimized for
use with GelRed™ and GelGreen™ precast gels. GelRed™ and
GelGreen™ also are compatible with popular gel extraction kits
and DNA ladders from other suppliers.
GelRed™, GelGreen™, and their uses are covered by granted US and/or international patents. SYBR® is registered trademark of Life Technologies
1 kb and 100 bp DNA ladders
• Ready-to-use at optimal
concentration for loading
GelRed™ and GelGreen™
precast gels
• 1 kb ladder: 250 bp-10 kb
• 100 bp ladder: 100 bp-1.5 kb
• Also available with loading
buffer supplied separately.
Figure 16. 100 ng each of 1 kb ladder
(left) and 100 bp ladder (right) run on
1% agarose/TBE gels containing 1X
GelRed™. Gel was imaged with an EtBr
filter using a GelDocIt™ system from UVP.
bp 10,000 & 8,000
250
500
750
1,000
1,500
2,000
2,500
3,000
4,000
6,000 & 5,000
bp 1,500
1,000
900
800
700
600
500
400
300
200
100
1 kb 100 bp
GelRedTM and GelGreenTM Nucleic Acid Gel Stains
Molecular Biology Products • www.biotium.com 11
0
250
500
750
1000
0 0.1 0.5 1 5 10 25 50
N
um be r o
f C
ol on ie s Dose (ug/plate)
SYBR Green
EtBr
GelGreen
GelRed
Safer, ultra-sensitive alternatives
to EtBr and SYBR® dyes
Figure 18. GelRed™ and GelGreen™ are non-mutagenic. Comparison of mutagenicity
among GelGreen™, GelRed™, SYBR® Green I and EtBr in +1 frameshift Salmonella
indicator strain TA98 in the presence of S9 fraction. SYBR® Green I became cytotoxic at
50 ug/plate. For more information, download the GelRed™ and GelGreen™ Safety Report
at www.biotium.com.
FEATURES
Safer than EtBr
Shown by Ames test and other tests to be
nonmutagenic and noncytotoxic.
Easy disposal
Passed environmental safety tests for direct
disposal down the drain or in regular trash.
Ultra-sensitive
More sensitive than EtBr and SYBR® Safe.
Extremely stable
Stable in solution at room temperature.
Simple to use
For precast or post-electrophoresis gel staining.
Compatible with standard instruments
GelRed™ replaces EtBr; GelGreen™ replaces
SYBR® dyes.
Description Cat. No. Unit Size
GelRed™ 10,000X in water 41003-T, 41003, 41003-1 25 uL, 0.5 mL, 10 mL
GelRed™ 10,000X in DMSO 41002, 41002-1 0.5 mL, 10 mL
GelRed™ 3X in water 41001 4 L
GelGreen™ 10,000X in water 41005-T, 41005, 41005-1 25 uL, 0.5 mL, 10 mL
GelGreen™ 10,000X in DMSO 41004 0.5 mL
6X GelRed™ Prestain Loading
Buffer with Blue Tracking Dyes 41009 1 mL
6X GelRed™ Prestain Loading
Buffer with Orange Tracking Dye 41010 1 mL
Ready-To-Use
1 kb DNA Ladder 31022 150 lanes
Ready-To-Use
100 bp DNA Ladder 31032 150 lanes
1 kb DNA Ladder 31039 30 ug
100 bp DNA Ladder 31040 30 ug
DNA Gel Extraction Kit 31030-50, 31030-250 50 columns, 250 columns
5X TBE Buffer 41006 4 L
Ordering Information
Figure 17. GelRed™ and GelGreen™ gel stains are safer because they cannot
penetrate cell membranes to bind DNA in living cells. HeLa cells were incubated
at 37oC with 1X SYBR® Safe, GelGreen™ or GelRed™, respectively. Images
were taken following incubation with dye for 30 min. The top row shows phase
contrast images of the field of cells, the bottom row shows green fluorescence for
SYBR® Safe and GelGreen™ and red fluorescence for GelRed™. SYBR® Safe
rapidly entered cells and stained nuclei. GelRed™ and GelGreen™ were unable
to cross cell membranes, demonstrated by the absence of fluorescence staining.
SYBR® Safe GelRed™ GelGreen™
* GelRed™ and GelGreen™ also are compatible with popular gel extraction kits
and DNA ladders from other suppliers.
References
Search GelRed Biotium or GelGreen Biotium on Google Scholar: GelRed™ has
been cited in more than 1500 publications, and GelGreen™ has been cited in
more than 100 publications to date.
Haines, et al. Properties of nucleic acid staining dyes used in gel electrophoresis.
Electrophoresis. Mar; 36(6), 941-4 (2015).
Ohta, et al. Ethidium bromide and SYBR Green I enhances the genotoxicity of UVirradiation and chemical mutagens in E. coli. Mut. Res 492, 91 (2001).
12 Molecular Biology Products • www.biotium.com
The safety and sensitivity of
GelRed™ and GelGreen™
now for PAGE gels
PAGE GelGreen™PAGE GelRed™SYBR® Safe
Figure 20. PAGE GelRed™ and PAGE GelGreen™ gel stains are safer because they cannot
penetrate cell membranes to bind DNA in living cells. HeLa cells were incubated at 37oC with
1X SYBR® Safe, 1X PAGE GelRed™, or 1X PAGE GelGreen™. Images were taken following
incubation with dye for 30 min using FITC filter set for SYBR® Safe and PAGE GelGreen™, and
Cy®3 filter set for PAGE GelRed™. SYBR® Safe rapidly penetrated cell membranes as evident
from the bright green staining of nuclei and cytoplasm. However, PAGE GelRed™ and PAGE
GelGreen™ were unable to cross cell membranes, as shown by the absence of fluorescence
staining in healthy cells. Staining was observed in dead cells present sporadically in the cultures,
as is observed with other non-membrane permeable nucleic acid dyes. The presence of cells in the
field of view was confirmed by phase contrast microscopy (not shown).
Figure 19. NEB low molecular weight ladder was separated
on a 10% acylamide TBE gel (left to right, 500, 200, 100 ng/
lane) and stained with 1X PAGE GelRed™ (left) or 1X PAGE
GelGreen™ (right) in water for 30 minutes. Gels were imaged
on a UV transilluminator using a UVP GelDoc-It imaging
system with ethidium bromide filter for PAGE GelRed™ or
SYBR® filter for PAGE GelGreen™, with 2 second exposure
time.
Biotium scientists recognize that a fundamental approach for making a gel stain safe is to eliminate or minimize the chance for the dye to
interact with genomic DNA in living cells. Based on this design principle,
chemists at Biotium incorporated structural features to make the dyes
impermeable to latex gloves, nitrile gloves, and cell membranes.
In the design of the original GelRed™ and GelGreen™ dyes, we
achieved the dyes’ membrane impermeability mainly by making the
dyes physically large. While this strategy works extremely well to
improve the dyes’ safety and at the same time produces exceptional
gel staining sensitivity for agarose gels, the relatively large size of
GelRed™ and GelGreen™ make the dyes difficult to penetrate into
the more densely packed polyacrylamide gels, rendering the dyes
less optimal for PAGE gel staining. In designing PAGE GelRed™ and
PAGE GelGreen™ dyes, we used a novel approach to make the dyes
membrane impermeable without making the dyes large. Importantly,
the new design strategy still ensures that the PAGE dyes possess
essential properties for gel staining, including good sensitivity, stability
and compatibility with existing instruments and downstream sample
analysis.
PAGE GelRed™ PAGE GelGreen™
Safer gel stains designed specifically for use in
polyacrylamide gels
 Formulated in water and impermeable to latex and
nitrile gloves
 Non-toxic and non-mutagenic in AMES test
 Non-toxic to aquatic life, OK for drain disposal by EPA
Title 22 hazardous waste test
Download the complete PAGE GelRed™ and PAGE
GelGreen™ Safety Report at www.biotium.com
PAGE GelRedTM and PAGE GelGreenTM
Designed specifically for polyacrylamide gel staining of nucleic acids
Description Cat. No. Unit Size
PAGE GelRed™ 10,000X in
water 41008-T, 41008-500 uL 0.1 mL, 0.5 mL
PAGE GelRed™ 1X in water 41014 4 L
PAGE GelGreen™ 10,000X
in water 41007-T, 41007-500 uL 0.1 mL, 0.5 mL
PAGE GelGreen™ 1X in water 41013 4 L
Ordering Information
PAGE GelRed™ and PAGE GelGreen™ are covered by pending patents.
Molecular Biology Products • www.biotium.com 13
Figure 21. A. Two-fold serial dilutions of Precision Plus Protein™ standards (Bio-Rad) were
separated via SDS-PAGE and then stained with LumiteinTM for 90 minutes without a separate
fixation step. Gels were imaged on a GE Typhoon Trio™ scanner using 532 nm excitation and
610 BP 30 emission filter. B. 2-D gel of human liver protein lysate stained with Lumitein™ and
imaged on a GE Typhoon Trio™ scanner. The three circled spots were excised and subjected
to mass spectroscopy (MS) analysis by Applied Biomics, Inc. (Hayward, CA). The results
confirmed that Lumitein™ gel staining is fully compatible with MS analysis (data not shown).
A B
FEATURES
Highly sensitive
At least as sensitive as silver stain, allowing detection of
as little as 1 ng protein or less (Figure 21A).
Simple & fast staining
Fixation and staining is combined in a single step, for
results in 30-90 minutes. Gels can be left in staining
solution longer than 90 minutes without overstaining.
Native gels can be stained after a brief soak in SDS/
acetic acid.
Compatible with common instruments
Image using a UV light box, Dark Reader®, or laser
scanner.
Wide linear detection range
At least three orders of magnitude.
Compatible with downstream analysis
Compatible with mass spectroscopy and Edman peptide
sequencing (Figure 21B).
Economical and convenient
Available as a 100X concentrated solution to reduce
manufacturing cost and shipping cost, or as a ready-touse 1X staining solution for convenience.
Highly stable
100X concentrate and 1X working solution are stable at
room temperature for at least 1 year.
Table 2. Comparison of standard staining protocols
Lumitein™ and its related technologies are covered by pending US and international patents. Lumitein™ is a trademark of Biotium, Inc. Dark Reader® is a registered trademark of Clare Chemical Research;
Typhoon Trio™ is a trademark of GE Healthcare; Precision Plus™ Protein is a trademark of Bio-Rad Laboratories; SYPRO® is a registered trademark of Life Technologies.
Protocol
Step SYPRO
® Ruby Lumitein™
Fixation
step 1
15 min. 50% methanol/
7.5% acetic acid None
Fixation
step 2
15 min. 50% methanol/
7.5% acetic acid None
Staining Overnight 30-90 min.
Destaining 30 min. 10% methanol/ 7% acetic acid
No destaining required
Optional: 5 min. in 30%
methanol/ 15% acetic acid or 20
min. in water
Rinse 1 5 min. water
Single 5 min. water rinse
Rinse 2 5 min. water
Total time More than 16 hours 2 hours or less
Description Cat. No. Size
Lumitein™ Protein Gel Stain, 1X
21001 200 mL
21001-1 1 L
21001-2 5 x 1 L
Lumitein™ Protein Gel Stain, 100X
21002 2 mL
21002-1 10 mL
21002-2 50 mL
Ordering Information
~ 0.2 ng
LumiteinTM Protein Gel Stain
Ultra Sensitive, Rapid PAGE Gel Protein Staining
14 Molecular Biology Products • www.biotium.com
PMATM and PMAxxTM for Viability PCR
Selectively amplify DNA from live bacteria by removing dead cell
DNA with PMATM and PMAxxTM dyes!
PMATM and PMAxxTM are membrane-impermeable dyes that enter
dead cells and modify their DNA, preventing PCR amplification.
Viability PCR using PMATM or PMAxxTM has many uses including:
• Food safety
• Probiotic detection
• Environmental testing
• Infectious disease testing
• Microbiology research
PMA™ is a photoreactive dye with a high affinity for DNA. The dye intercalates into dsDNA and forms a covalent linkage upon exposure to intense visible light. PMA™ inhibits PCR amplification
of modified DNA templates by a combination of removal of modified DNA during purification and
inhibition of template amplification by DNA polymerases.
PMAxx™ is a next-generation, improved version of PMA™ developed at Biotium. It works in a similar manner as PMA™, but has been optimized for less penetration into live cells and higher activity in dead cells, resulting in better discrimination between live and dead cells.
Pre-treating a bacterial sample with PMA™ prior to PCR analysis permits one to selectively detect viable bacteria in a highly sensitive and
reliable manner. Because PMA™ is designed to be cell membrane-impermeable, when a sample containing both live and dead bacteria is
treated with PMA™, only dead bacteria with compromised cell membranes are susceptible to DNA modification. In a real-time PCR reaction,
dead cell DNA will show delayed amplification and higher Ct than live cells. In a mixed population, qPCR permits quantitation of cell viability.
The PMA™-qPCR technology can be applied not only to bacteria but to other cell types as well.
PMA™-PCR has been reported for a wide variety of microorganisms in numerous publications; visit www.biotium.com to download a reference
list.
Figure 22. The cell membrane-impermeable PMA™ dye selectively and covalently modifies DNA
from dead bacteria with compromised membrane while leaving DNA from viable cells intact.
Because PMA™-modified DNA can not be amplified, subsequent lysis of viable cells and qPCR
permit selective quantitation of viable bacteria.
Figure 23. Effect of PMATM on qPCR of DNA from live and heat-inactivated E. coli. qPCR was performed using primers against a region of the 16S rRNA gene. (A) Representative
amplification curves for real-time PCR performed on DNA from PMATM-treated live and heat-killed E. coli. (B) The dCt of live and killed E. coli with and without PMATM treatment. The Ct value of
sample without PMATM was subtracted from the corresponding sample with PMATM cross-linking (Ct with PMATM – Ct without PMATM).
A B
PMAxxTM and its uses are covered by pending US and/or international patents.
Molecular Biology Products • www.biotium.com 15
PMA™ Enhancer for Gram-Negative Bacteria
Under some conditions such as mild heat treatment, bacteria may
be dead but retain intact membranes that have lower permeability
to PMA™ than those of boiled bacteria, for example. This could
result in an overestimate of live bacteria. Biotium has developed
an Enhancer for use with Gram-Negative bacteria that can greatly
improve live/dead discrimination.
PMA™ Real-Time PCR Bacterial Viability Kits
PMA™-PCR kits are designed for selective detection of viable
bacteria from a specific strain using PMA™ dye and real-time
PCR. We offer kits for detection of selected strains of bacteria that
are of widespread interest to food safety, public health, and/or
antibacterial research. More strains are being added, so check our
website for current offerings.
Kits include:
• PMA™ dye
• Fast EvaGreen® qPCR Master Mix
• ROX reference dye
• Validated PCR primers for specific bacterial strain
• 5X PMA™ Enhancer for Gram-Negative Bacteria (gramnegative strains only)
Kits available for:
• Salmonella enterica
• Staphylococcus aureus
• MRSA
• Escherichia coli and Escherichia coli O157:H7
• Mycobacterium tuberculosis
• Listeria monocytogenes
PMA-Lite™ LED Photolysis Device
PMA-Lite™ is specifically designed for photoactivation of PMA™ and other similar
dyes. Receive a free vial of PMA™ dye when you purchase a PMA-Lite™ LED
Photolysis Device.
Features:
• Provides even illumination to up to 18 microcentrifuge tubes.
• Internal fan to ensure a temperature of <37OC.
• Four timer settings for 10, 15, 20 or 30 minutes of illumination.
• Long-lasting LEDs with 465-475 nm emission for efficient activation of PMA™,
EMA or other similar azido dyes.
0
2
4
6
8
10
12
control enhancer
dC t(P
MA
-n oP MA
)
Change in Ct values after PMA treatment
Figure 24. PMA™ plus Enhancer for quantitation of viable bacteria by Real-time
PCR. A. Mildly heat-killed E. coli were treated with PMA™ and/or Enhancer, followed
by exposure with the PMA-Lite™ and DNA purification. dCt values were calculated by
subtracting the Ct without PMA™ from the Ct with PMA™. For dead cells, the use of
Enhancer increased the dCt from 4 to 10, greatly increasing the specificity of viability
PMA™-PCR.
PMATM and PMAxxTM for Viability PCR
Cat. # Product Name Unit Size
40013 PMA™ dye 1 mg
40019 PMA™ dye, 20 mM in dH2O 100 uL
40069 PMAxx™ dye, 20 mM in dH2O 100 uL
E90002 PMA-Lite™ LED Photolysis Device 1 device
31038 PMA™ Enhancer for Gram-Negative Bacteria 16 mL
31033 PMA™-PCR Bacterial Viability Kit-Salmonella 200 assays
31034 PMA™-PCR Bacterial Viability Kit-M. tuberculosisis 200 assays
31035 PMA™-PCR Bacterial Viability Kit-Staph. aureus 200 assays
31036 PMA™-PCR Bacterial Viability Kit-MRSA 200 assays
31037 PMA™-PCR Bacterial Viability Kit-E. coli O157:H7 200 assays
31050 PMA™-PCR Bacterial Viability Kit-E. coli 200 assays
31051 PMA™-PCR Bacterial Viability Kit-Listeria 200 assays
Ordering Information
Biotium, Inc.
www.biotium.com
800-304-5357
General Inquiries
btinfo@biotium.com
Quotes and Ordering
order@biotium.com
Technical Support
techsupport@biotium.com
www.biotium.com
Glowing Products for Science™

PDF Document reader online

This website is focused on providing document in readable format, online without need to install any type of software on your computer. If you are using thin client, or are not allowed to install document reader of particular type, this application may come in hand for you. Simply upload your document, and Docureader.top will transform it into readable format in a few seconds. Why choose Docureader.top?

  1. Unlimited sharing - you can upload document of any size. If we are able to convert it into readable format, you have it here - saved for later or immediate reading
  2. Cross-platform - no compromised when reading your document. We support most of modern browers without the need of installing any of external plugins. If your device can oper a browser - then you can read any document on it
  3. Simple uploading - no need to register. Just enter your email, title of document and select the file, we do the rest. Once the document is ready for you, you will receive automatic email from us.

Previous 10

Next 10