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Archive for January, 2012

Real Time PCR Dyes

Dye Structure Color Vendor Cat # Price Features
SYBR Green 1 Green unstable at 4 °C
SYTO-82 Orange does not inhibit PCR, show no preferential binding to GC rich sequences and does not influence melting temperature, Tm, even at high concentrations
SYTO-13 Green does not inhibit PCR, show no preferential binding to GC rich sequences and does not influence melting temperature, Tm, even at high concentrations
SYTO-9 Green produces highly reproducible DNA melting curves over a broader range of dye concentrations, low PCR inhibition
SYTO-16 melting curve analysis: produces the sharpest peaks.
SYTO-80 Orange
EvaGreen Green melting curve analysis: produces the sharpest peaks.
BEBO 468/492 TATAA BiocenterChalmers University of Technology
BOXTO 515/552 TATAA BiocenterChalmers University of Technology
BETIBO 468/492
BOXTO-MEE Chalmers University of Technology better stability, 45 days storage at 4 °C. better than BOXTO, BOXTO-PRO, BEBO, BETIBO and SYBR Green 1
Dye Concentration R2 Slope Efficiency
SYBR Green 0.32 × (0.64 μM) 0.985 -3.40 97%
SYBR Green 1× (2 μM) 0.395 -2.13 195%
EvaGreen 1× 0.995 -3.30 101%
EvaGreen 5× 0.999 -3.46 95%
SYTO 13 5 μM 0.993 -3.42 96%
SYTO 13 10 μM 0.993 -3.44 95%
SYTO-16 5 μM 0.983 -3.30 101%
SYTO-16 10 μM 0.992 -3.43 96%
SYTO 80 10 μM 0.992 -3.42 96%
SYTO 80 20 μM 0.986 -3.41 96%
SYTO 82 10 μM 0.998 -3.41 96%
SYTO 82 20 μM 0.996 -3.37 98%

Comments

January 30, 2012 at 11:47 am ·

DNA Stain Structure

BEBO

BEBO

BEBO

BEBO_BETO_BOXTO

BETIBO

BOXBO BEBO BETO

BOXTO

BOXTO BETO

BOXTO-MEE

BOXTO-PRO

Cyan46 Cyan2

DNA_bound_cyanine_dimers

GelGreen_GelRed

Invitrogen_dyes

RO-PRO, Bo-PRO, YO-PRO, TO-PRO

SYTO

SYBR GREEN I

SYBR Green 1 & Pico Green

TO

TO

TOAC

TOPY

TOPY

TOTO-1 vs PicoGreen

TOTO-3

TOTO YOYO

YO

YOYO

Comments

January 29, 2012 at 4:36 pm ·

Nucleic Acid Stains

Name of Dye Ex/Em (in nm) Specificity Sensitivity Factors Afftecting Performance Advantages Disadvantages
DAPI 360/465 A-T Detergents,
polyphosphates,dextran sulfate		 inexpensive, strongly A-T selective
Hoechst 33258 360/465 A-T of dsDNA 10 ng/mL pH, SDS inexpensive, sensitive strongly A-T selective
PicoGreen 485/535 all dsDNA 250 pg/mL none demonstrated simple, sensitive, no
reactioncleanup necessary
 		 cost
RiboGreen 485/535 G of all nucleic acids 200 pg/mL presence of any DNA quantitation of RNA with
simpleDNase treatement
OliGreen 485/535 T of all nucleic acids 100 pg/mL presence of dsDNA or RNA quantitation of ssDNA,sensitivity will dectect dsDNA and RNApresent
cyanine dyes (e.g. YOYO-1) varies dsDNA, some single-
stranded oligionucleotides		 0.5 to 2.5 ng/mL salt, ethanol, SDS; some
variantsshow base selectivity		 inexpensive, relatively sensitive less sensitive or selective than
PicoGreen,
double bands: TOTO-1, YOYO-1, ethidium homodimer-1 in agarose gel
ethidium bromide 520/605 dsDNA and RNA 2 ng (per band, agarose gel) degrades in the presence
ofsodium nitrate and hypophosphorous acid, must be stored in the
absenceof light		 very inexpensive, goodsensitivity toxic mutagen with possible
carcinogenic properties as well, not used with microplate readers
Sybr Green I 485/535 dsDNA 25 pg (per band,
agarose gel)		 must be stored in the absence
oflight		 inexpensive dye for use
in qPCR reactions, not mutagenic		 not commonly used with
microplate readers
GelGreen 300(500)/~520 dsDNA 25 pg (per band,
agarose gel)		 Nonmutagenic and noncytotoxic
Safe to aquatic life for direct disposal in the drain
Suitable for staining either DNA or RNA
Much more sensitive than EtBr
No need to change the instrument setting used for EtBr
Perfectly stable at room temperature
GelRed 300(595)/~600 dsDNA 25 pg (per band,
agarose gel)		 Nonmutagenic and noncytotoxic
Safe to aquatic life for direct disposal in the drain
Suitable for staining either DNA or RNA
Much more sensitive than EtBr
No need to change the instrument setting used for EtBr
Perfectly stable at room temperature
Sybe Safe 280,502/530 dsDNA 1 ng (per band,
agarose gel)
SYBR Gold 300, 495/ dsDNA 20 pg (per band,
agarose gel)
GelStar (Lonza,
Cambrex)		 493/527 dsDNA 20 pg of dsDNA, 3 ng of RNA
Vistra Green
(GE Amershan)		 490/520
488/532
 		 dsDNA 3 pg (per band,
agarose gel)
BOXTO DNA-dye complex: slow and
salt‐insensitive dissociation

Comments

January 29, 2012 at 3:04 pm ·

Nucleic acid Stains

Probe Ex (nm) Em (nm) MW Notes
Hoechst 33342 343 483 616 AT-selective
DAPI 345 455 AT-selective
Hoechst 33258 345 478 624 AT-selective
SYTOX Blue 431 480 ~400 DNA
Chromomycin A3 445 575 CG-selective
Mithramycin 445 575
YOYO-1 491 509 1271
Ethidium Bromide 493 620 394
Acridine Orange 503 530/640 DNA/RNA
SYTOX Green 504 523 ~600 DNA
TOTO-1, TO-PRO-1 509 533 Vital stain, TOTO: Cyanine Dimer
TO-PRO: Cyanine Monomer
Thiazole Orange 510 530
Propidium Iodide (PI) 536 617 668.4
LDS 751 543;590 712;607 472 DNA (543ex/712em), RNA (590ex/607em)
7-AAD 546 647 7-aminoactinomycin D, CG-selective
SYTOX Orange 547 570 ~500 DNA
TOTO-3, TO-PRO-3 642 661
DRAQ5 647 681/697 413 (Biostatus) (usable excitation down to 488)
Nucleic acid probes
Hoechst 33342 343 483 616 AT-selective
DAPI 345 455 AT-selective
Hoechst 33258 345 478 624 AT-selective
SYTOX Blue 431 480 ~400 DNA
Chromomycin A3 445 575 CG-selective
Mithramycin 445 575
YOYO-1 491 509 1271
Ethidium Bromide 493 620 394
Acridine Orange 503 530/640 DNA/RNA
SYTOX Green 504 523 ~600 DNA
TOTO-1, TO-PRO-1 509 533 Vital stain, TOTO: Cyanine DimerTO-PRO: Cyanine Monomer
Thiazole Orange 510 530
Propidium Iodide (PI) 536 617 668.4
LDS 751 543;590 712;607 472 DNA (543ex/712em), RNA
(590ex/607em)
7-AAD 546 647 7-aminoactinomycin D, CG-selective
SYTOX Orange 547 570 ~500 DNA
TOTO-3, TO-PRO-3 642 661
DRAQ5 647 681,697 413 (Biostatus)
(usable excitation down to 488)
DNA Dye Abs. Max. (nm) Em. Max. (nm) ε (cm-1) Color of Fluorscence
YOYO-1 491 509 99,000 Green
TOTO-1 514 533 117,000 Green
DAPI 358 461 24,000 Blue
Nuclear Yellow 355 495 36,000 Yellow
Hoechst 33342 350 461 45,000 Blue
BOBO-3 570 604 148,000 Orange
YOYO-3 612 631 167,000 Orange

Comments

January 29, 2012 at 12:03 am ·

classic nucleic acid stains

Cat # Dye Ex/Em * Fluorescence Emission Color Applications †
A666 Acridine homodimer 431/498 Green
  • Impermeant
  • AT-selective
  • High-affinity DNA binding
A1301

A3568 ‡

 Acridine orange

500/526 (DNA)

460/650 (RNA)

 Green/Red
  • Permeant
  • RNA/DNA discrimination measurements
  • Lysosome labeling
  • Flow cytometry
  • Cell-cycle studies
A1310 7-AAD (7-amino-actinomycin D) 546/647 Red
  • Weakly permeant
  • GC-selective
  • Flow cytometry
  • Chromosome banding
A7592 Actinomycin D 442 None
  • Chromosome banding
A1324 ACMA 419/483 Blue
  • AT-selective
  • Alternative to quinacrine for chromosome Q banding
  • Membrane phenomena
D1306

D3571

D21490

 DAPI 358/461 Blue
  • Semi-permeant
  • AT-selective
  • Cell-cycle studies
  • Mycoplasma detection
  • Chromosome and nuclei counterstain
  • Chromosome banding
D1168

D11347

D23107

 Dihydroethidium 518/605 Red §
  • Permeant
  • Blue fluorescent until oxidized to ethidium
15585-011 Ethidium bromide 518/605 Red
  • Impermeant
  • dsDNA intercalator
  • Dead-cell stain
  • Chromosome counterstain
  • Electrophoresis
  • Flow cytometry
  • Argon-ion laser excitable
E1169 Ethidium homodimer-1 (EthD-1) 528/617 Red
  • Impermeant
  • High-affinity DNA labeling
  • Dead-cell stain
  • Electrophoresis prestain
  • Argon-ion and green He-Ne laser excitable
E3599 Ethidium homodimer-2 (EthD-2) 535/624 Red
  • Impermeant
  • Very high-affinity DNA labeling
  • Electrophoresis prestain
E1374 Ethidium monoazide 464/625

(unbound)**

 Red
  • Impermeant
  • Photocrosslinkable
  • Compatible with fixation procedures
H7593 Hexidium iodide 518/600 Red
  • Permeant, except gram-negative bacteria
  • Stains nuclei and cytoplasm of eukaryotes and some bacteria
H1398

H3569 ‡

H21491

 Hoechst 33258 (bis-benzimide) 352/461 Blue
  • Permeant
  • AT-selective
  • Minor groove–binding
  • dsDNA-selective binding
  • Cell-cycle studies
  • Chromosome and nuclear counterstain
H1399

H3570 ‡

H21492

 Hoechst 33342 350/461 Blue
  • Permeant
  • AT-selective
  • Minor groove–binding
  • dsDNA-selective binding
  • Cell-cycle studies
  • Chromosome and nuclear counterstain
H21486 Hoechst 34580 392/498 Blue
  • Permeant
  • AT-selective
  • Minor groove–binding
  • dsDNA-selective binding
  • Cell-cycle studies
  • Chromosome and nuclear counterstain
H22845 Hydroxystilbamidine 385/emission varies with nucleic acid Varies
  • AT-selective
  • Spectra dependent on secondary structure and sequence
  • RNA/DNA discrimination
  • Nuclear stain in tissue
L7595 LDS 751

543/712 (DNA)

590/607 (RNA)

 Red/infrared
  • Permeant
  • High Stokes shift
  • Long-wavelength spectra
  • Flow cytometry
N21485 Nuclear yellow 355/495 Yellow
  • Impermeant
  • Nuclear counterstain
P1304MP

P3566 ‡

P21493

 Propidium iodide (PI) 530/625 Red
  • Impermeant
  • Dead-cell stain
  • Chromosome and nuclear counterstain

* Excitation (Ex) and emission (Em) maxima in nm.
All excitation and emission maxima were determined for dyes bound to double-stranded calf thymus DNA in aqueous solution, unless otherwise indicated.

† Indication of dyes as “permeant” or “impermeant” are for the most common applications; permeability to cell membranes may vary considerably with the cell type, dye concentrations and other staining conditions.

‡ Available in aqueous solution for those wishing to avoid potentially hazardous and mutagenic powders. § After oxidation to ethidium. ** Prior to photolysis; after photolysis the spectra of the dye/DNA complexes are similar to those of ethidium bromide–DNA complexes.

Comments

January 28, 2012 at 11:33 pm ·

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