Detection and Sizingof DNA in Agarose Gels

Section IV: Detection and Sizing of DNA in Agarose Gels

In This Section
Guide to Lonza Ladders and Markers
Detecting DNA with GelStar® and SYBR® Green
Nucleic Acid Gel Stains
Detecting DNA with Ethidium Bromide
High Sensitivity Detection using theFlashGel® System
References

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Section IV: Detection and Sizing of DNA in Agarose Gels
Guide to Lonza Ladders and Markers

Size Range (Bold numbers indicate brighter bands)
20 bp
Standard Ladders(Cat. No.)
50330
SimplyLoad® Ladders(Cat. No.)
50331
Number of25
Fragments
Size Range 20 bp –500 bp500 bp
480 bp460 bp440 bp420 bp400 bp380 bp360 bp340 bp320 bp300 bp280 bp260 bp240 bp220 bp200 bp180 bp160 bp140 bp120 bp100 bp
80 bp
60 bp
40 bp
20 bp

20 bp Ext

100 bp100 bpext

Tandem

500 bpQuant
Ladder

Rev QuantLadder

50 bp1000 bp

50 bp2500 bp

1 kb10 kb

50320

50321

50322

NA

50323

50334

50335

50461

50631

50471

50326

50327

50328

50333

50329

50336

50337

NA

NA

NA

Detection and Sizingof DNA in Agarose Gels

50

10

30

21

16

5

5

9

13

9

20 bp –
1,000 bp1,000 bp
980 bp960 bp940 bp920 bp900 bp880 bp860 bp840 bp820 bp800 bp780 bp760 bp740 bp720 bp700 bp
680 bp660 bp640 bp620 bp600 bp580 bp560 bp540 bp520 bp500 bp
480 bp460 bp440 bp420 bp400 bp380 bp360 bp340 bp320 bp300 bp280 bp260 bp240 bp220 bp200 bp180 bp160 bp140 bp120 bp100 bp
80 bp
60 bp
40 bp
20 bp

100 bp –1,000 bp1,000 bp
900 bp800 bp700 bp600 bp500 bp400 bp300 bp200 bp100 bp10 bp –
3,000 bp3,000 bp
2,900 bp2,800 bp2,700 bp2,600 bp
2,500 bp
2,400 bp2,300 bp2,200 bp2,100 bp2,000 bp
1,900 bp1,800 bp
1,700 bp1,600 bp
1,500 bp
1,400 bp
1,300 bp1,200 bp
1,100 bp1,000 bp
900 bp800 bp700 bp600 bp500 bp400 bp300 bp200 bp100 bp100 bp –
12 kb
12 kb
11 kb
10 kb
9 kb
8 kb
7 kb
6 kb
5 kb
4 kb
3 kb
2 kb
1 kb
900 bp800 bp700 bp600 bp500 bp400 bp300 bp200 bp100 bp500 bp –8 kb
8,000 bp
7,500 bp7,000 bp
6,500 bp6,000 bp
5,500 bp5,000 bp
4,500 bp4,000 bp
3,500 bp3,000 bp
2,500 bp2,000 bp
1,500 bp1,000 bp
500 bp100 bp –1,000 bp1,000 bp
700 bp500 bp200 bp100 bp100 bp –1,000 bp1,000 bp
700 bp500 bp200 bp100 bp50 bp –
1,000 bp1,000 bp
700 bp525 bp500 bp400 bp300 bp200 bp100 bp
50 bp

50 bp –
2,500 bp
2.5 kb2 kb
1.5 kb1250 bp
1 kb
700 bp525 bp500 bp400 bp300 bp200 bp100 bp
50 bp

1 kb –
10 kb
10 kb
7 kb
5 kb
4 kb
3 kb
2.5 kb2 kb
1.5 kb1 kb

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Section IV: Detection and Sizing of DNA in Agarose Gels
Guide to Lonza Ladders and Markers — continued

FlashGel® DNA and RNA Markers
Size Range

0.5 Kb - 9.0 kb

0.5 Kb - 9.0 kb

100 bp - 3 kb

100 bp - 4 kb

100 bp - 1.5 kb

50 bp - 1.5 kb

Ladder/Marker

RNA Marker

RNA Marker

DNA Marker

DNA Marker

QuantLadder

DNA Marker

50575

50577

57034

50473

50475

57033

10

6

5

7

5

8

9.0 kb

9.0 kb3,000 bp4,000 bp

1,500 bp

1,500 bp
800 bpCat. No.
Number of Bands

6.0 kb

5.0 kb1,500 bp2,000 bp

800 bp5.0 kb

3.0 kb800 bp1,250 bp

400 bp500 bp

4.0 kb

1.5 kb

500 bp800 bp250 bp300 bp3.0 kb

1.0 kb100 bp300 bp100 bp200 bp2.5 kb

0.5 kb

2.0 kb200 bp150 bp100 bp100 bp1.5 kb50 bp

1.0 kb
0.5 kbFlashGel® System Markers and Ladders
Catalog No.

50473

50475

57034

57033

– 4,000 bp– 2,000 bp– 1,250 bp– 800 bp

– 1,500 bp (30ng)– 1,500 bp– 800 bp
– 500 bp50577
– 3,000 bp– 1,500 bp– 800 bp
– 500 bp
– 300 bp– 100 bp

– 800 bp (21ng)– 300 bp

– 400 bp (15 ng)– 200 bp

– 250 bp (7.5 ng)– 100 bp– 100 bp (3 ng)– 9 kb
– 9.0 Kb

– 5.0 Kb

– 1.0 Kb

– 150 bp

– 50 bp– 3 kb
– 2.5 kb

– 1.5 Kb

– 200 bp– 100 bp

– 6 kb– 5 kb– 4 kb

– 0.3 Kb

– 300 bp

– 500 bp50575

– 3,000 bp– 1,500 bp– 800 bp
– 500 bp
– 300 bp

– 0.5 Kb

– 2 kb
– 1.5 kb
– 1 kb

– 0.5 kb

– 100 bp

FlashGel® DNA Marker
100 bp–4 kb
1.2%, 12 + 1(Standard)FlashGel® DNA Marker
100 bp–1.5 kb
1.2%, 12 + 1(Quantitation)

FlashGel® DNA Marker
50 bp–1.5 kb
2.2%, 12 + 1(High Concentration)

FlashGel® DNA Marker
100 bp–3 kb
1.2%, 16 + 1(Double-tier)FlashGel® RNA Marker
0.5 kb–9 kb
1.2%, 12 + 1 & 16 +1(RNA)

Lonza RNA Marker
0.5 bp–9 kb
1.2%, 12 + 1(RNA)

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Section IV: Detection and Sizing of DNA in Agarose Gels
Detecting DNA with GelStar®, SYBR® Green I or II Nucleic Acid Gel Stains

Introduction
GelStar® and SYBR® Green Nucleic Acid Gel Stains arehighly sensitive fluorescent stains for detecting nucleic
acids in agarose gels. Unlike ethidium bromide, these stains
fluoresce only upon binding to nucleic acids. Backgroundstaining is minimal and destaining of gels is not required.
GelStar® Stain gives high detection sensitivity for doublestranded or single-stranded DNA and RNA. SYBR® GreenI Stain exhibits a preferential affinity for double-stranded
nucleic acids and SYBR® Green II Stain is most sensitive
for single-stranded nucleic acids.
Advantages–– Clear polypropylene container (e.g., Rubbermaid®
Recycling #5 Plastics)–– GelStar® Photographic Filter (Wratten® #9
equivalent) or SYBR® Green Photographic Filter(Wratten® #15 equivalent)–– Microcentrifuge–– UV transilluminator, Dark Reader® Transilluminator(Clare Chemical Research, Inc.) or CCD imaging
system■■ ReagentsHigh sensitivity – 25 - 100 times more sensitive than ethidium
bromideFlexible – Add GelStar® Stain directly to the agarose
solution or post-stain your gel with GelStar® or SYBR®
Green Stains
Fast – No destaining required
The table below compares the detection sensitivities
of several commonly used staining methods. Samples
detected with SYBR® Green Stains were post-stainedwith the dye. Samples detected with Ethidium Bromide
or GelStar® Stain were detected by in-gel staining.
Detection limits were determined by using DNA samples
that were serially diluted and by recording the lowest
amount that gave a visible band on photographs.
Photographic conditions were varied as needed to use
the longest exposure time possible without increasing gel
background to an unacceptable level.
Stain and Method
GelStar® Stain – in gel
Ethidium bromide, no destain
Ethidium bromide, destain
SYBR® Green I or II Stain■■ Materials

ssDNA
25 pg
1.25 ng
350 pg
60 pg

dsDNA
20 pg
350 pg100 pg20 - 30 pg–– Buffer between pH 7.5 - 8.5 (TAE, TBE or TE)–– GelStar® or SYBR® Green I or II Stain stock solution
Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

The photographs below demonstrate the detection
sensitivity of GelStar® Nucleic Acid Gel Stain when used
in-gel or when gels have been post-stained.

GelStar® Stain
In-GelPost-Stained

Lonza’s 500 bp DNA Ladder
was separated on 1% SeaKem®
GTG® Agarose gels 20 cm long,
4 mm thick, run in 1X TBE
Buffer (Prepared from Lonza’s
AccuGENE® 10X TBE Buffer) at 6
V/cm for 3 hours. GelStar® Stain
was diluted 1:10,000 and added
directly to the agarose or the gel
was post stained for 30 minutes
in a 1:10,000 dilution of GelStar®
Stain in buffer. Lane 1: 10 ng DNA/
band; Lane 2: 5 ng DNA/band;
Lane 3: 2.5 ng DNA/band; Lane 4:
1.25 ng DNA/band.

Detection and Sizingof DNA in Agarose Gels

The FlashGel® System includes gel cassettes pre-stainedwith a similar high sensitivity stain. Refer to page 18-25.

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Section IV: Detection and Sizing of DNA in Agarose Gels
Detecting DNA with GelStar®, SYBR® Green I or II Nucleic Acid Gel Stains — continued

Tips for staining gels with GelStar® or SYBR® Green I Stain
Follow the guidelines below to increase the detection
sensitivity of GelStar® or SYBR® Green Stains—— New clear polypropylene containers (e.g., Rubbermaid®
Recycling #5 Plastics) should be obtained for use with
GelStar® and SYBR® Green Stains; when stored in thedark in polypropylene containers, the diluted stain canbe used for up to 24 hours and will stain 2 to 3 gels withlittle decrease in sensitivity; the containers should be
rinsed with distilled water (do not use detergents)
after each use and dedicated to GelStar® or SYBR®
Green Stain use only—— These stains bind to glass and some non-polypropylene(polystyrene) plastics resulting in reduced or no signal
from the nucleic acid—— A 1X working solution of GelStar® or SYBR® Green Stain
should be prepared just prior to use from the 10,000X
stock solution by diluting in a pH 7.5 to 8.5 buffer (e.g.,
TAE, TBE or TE)

Procedure for staining DNA with GelStar® or
SYBR® Green Stains
For optimal resolution, sharpest bands and lowest
background, stain the gel with GelStar® or SYBR® Green
Stain following electrophoresis. Alternatively, GelStar®
Stain can be included in the agarose gel.
It is not recommended to include SYBR® Green Stains in
the agarose gel. When the dye is incorporated into the
agarose, the gel is more sensitive to DNA overloading, andthe electrophoretic separation of DNA may not be identicalto that achieved with ethidium bromide.
The photograph below demonstrates the effect of adding
SYBR® Green I Stain to the agarose.

SYBR® Green Gel Stain
In-Gel

1 2 3 4 5 6

7 8

Post-Stained

1 2 3 4 5 6

7 8—— Agarose gels should be cast no thicker than 4 mm.
As gel thickness increases, diffusion of the stain into
the gel is decreased, lowering the efficiency of DNA
detection—— Optimal sensitivity for GelStar® and SYBR® Green Stains
is obtained by using the appropriate photographic
filters for each stain—— GelStar® Stain: Wratten® or Tiffen® #9 Filter—— SYBR® Green Stains: Wratten® or Tiffen® #15 Filter—— We do not recommend photographing gels with a 254
nm transilluminator; an outline of the UV light source
may appear in photographs; a filter that will allow a
525 nm transmission and exclude infrared light isrequired

DNA markers were separated on 1% SeaKem® Gold Agarose gels
10-cm-long, 3 mm thick, run in 1X TBE Buffer for 1 - 1.5 hours. SYBR®
Green I Stain was diluted 1:10,000 and added directly to the agaroseor the gel was post stained for 30 minutes in a 1:10,000 dilution of
SYBR® Green Stain in Buffer. Lanes 1 - 4: BstE II digest of lambda DNA;
1:2 dilutions with an initial loading of 25 ng/lane. Lane 5: 1 kb DNA
ladder (Invitrogen, Inc.); 200 ng/lane (SYBR® Green I stained gel); 1µg/lane (Ethidium bromide stained gel). Lane 6: Lonza’s 1-10 kb DNA
Ladder; 100 ng/lane. Lane 7: Gensura’s 1 kb DNA Ladder; 100 ng/lane.
Lane 8: Lonza’s 100 bp DNA Ladder; 100 ng/lane.

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Section IV: Detection and Sizing of DNA in Agarose Gels
Detecting DNA with GelStar®, SYBR® Green I or II Nucleic Acid Gel Stains — continued

Follow the steps below to stain DNA after electrophoresis
1.

Remove the concentrated stock solution of GelStar®
or SYBR® Green Stain from the freezer and allow thesolution to thaw at room temperature.

2.

Spin the solution in a microcentrifuge to collect the dye atthe bottom of the tube.

3.

Dilute the 10,000X concentrate to a 1X working solution(1 µl per 10 ml), in a pH 7.5 - 8.5 buffer, in a clear plastic
polypropylene container. Prepare enough staining
solution to just cover the top of the gel.

4.

Remove the gel from the electrophoresis chamber.

5.

Place the gel in staining solution.

6.

Gently agitate the gel at room temperature.

7.
Stain the gel for 15 - 30 minutes.
	The optimal staining time depends on the thickness ofthe gel, concentration of the agarose, and the fragment
size to be detected. Longer staining times are required
as gel thickness and agarose concentration increase.

9.

Remove the gel from the electrophoresis chamber.

10.

View with a 300 nm UV transilluminator, CCD camera or
Dark Reader® Transilluminator (Clare Chemical Research,Inc.). GelStar® Stained gels do not require destaining.
The dye’s fluorescence yield is much greater whenbound to DNA than when in solution.

Staining vertical gels with GelStar® andSYBR® Green Stains
Incorporating GelStar® and SYBR® Green Stains into thegel or prestaining the DNA for use in a vertical format is
not recommended. The dye binds to glass or plastic plates
and DNA may show little to no signal. Gels should be poststained as described in the previous section.
Follow this procedure when staining vertical gels with
GelStar® or SYBR® Green Stain
1.

Remove the concentrated stock solution of GelStar®
or SYBR® Green Stain from the freezer and allow thesolution to thaw at room temperature.

2.

Spin the solution in a microcentrifuge to collect the dyeat the bottom of the tube.

3.

Dilute the 10,000X concentrate to a 1X working solution,
in a pH 7.5 - 8.5 buffer, in a clear plastic polypropylene
container. Prepare enough staining solution to just cover
the top of the gel.

4.

Remove the gel from the electrophoresis chamber.

5.

Open the cassette and leave the gel in place on one
plate.

Remove the concentrated stock solution of GelStar®
Stain from the freezer and allow the solution to thaw.

6.

Place the plate, gel side up in a staining container.

7.

Gently pour the stain over the surface of the gel.

2.

Spin the solution in a microcentrifuge tube.

8.

Stain the gel for 5 - 15 minutes.

3.

Prepare the agarose solution (see pages 83-84).

9.

4.

Once the agarose solution has cooled to 70°C, add thestain by diluting the stock 1:10,000 into the gel solution
prior to pouring the gel (1 µl per 10 ml).

5.

Slowly swirl the solution.

View with a 300 nm UV transilluminator, CCD camera or
Dark Reader® Transilluminator (Clare Chemical Research,Inc.). GelStar® or SYBR® Green Stained gels do not require
destaining. The dye’s fluorescence yield is much greater
when bound to DNA than when in solution.

6.

Pour the gel into the casting tray (see page 85).

7.

Load your DNA onto the gel.

8.

Run the gel.

8.

Remove the gel from the staining solution and view with a
300 nm UV transilluminator, CCD camera or Dark Reader®
Transilluminator (Clare Chemical Research, Inc.).
GelStar® and SYBR® Green Stained Gels do not require
destaining. The dyes fluorescence yield is much greater
when bound to DNA than when in solution.

Follow this procedure when including GelStar® Stain in
the agarose gel.
1.

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Section IV: Detection and Sizing of DNA in Agarose Gels
Detecting DNA with GelStar®, SYBR® Green I or II Nucleic Acid Gel Stains — continued

Visualization by Photography

Application notes

Gels stained with GelStar® and SYBR® Green Stains exhibit
negligible background fluorescence, allowing long film
exposures when detecting small amounts of DNA. Use
the appropriate photographic filter for the stain you areusing.

—— The fluorescent characteristics of GelStar® and SYBR®
Green Stains make them compatible with argon ion
lasers.

The table below provides suggested film types andphotographic conditions
Polaroid® Film
Type 57 or 667
Type 55

f-stop4.5
4.5

Exposure time
0.5 - 2 seconds
15 - 45 seconds

Visualization by image capture system
For the best results and optimal sensitivity, visualize
GelStar® Stained Gels on The Dark Reader® Transilluminator(Clare Chemical Research, Inc.) GelStar® and SYBR® Green
Stains are compatible with most CCD and video imaging
systems. Due to variations in the filters for these systems,
you may need to purchase a new filter. Lonza does notsell filters for this type of camera. Contact your systems
manufacturer and using the excitation and emission
information listed, they can guide you to an appropriate
filter.
Stain
Emission (nm)GelStar® Stain
527
SYBR® Green I Stain
521
SYBR® Green II Stain
513

Excitation (nm)
493
494
497

—— These stains are removed from double-stranded DNA
by standard procedures for ethanol precipitation of
nucleic acids.
—— Gels previously stained with ethidium bromide cansubsequently be stained with GelStar® or SYBR® Green
Stain following the standard protocol for post-staining.
There will be some decrease in sensitivity whencompared to a gel stained with only GelStar® or SYBR®
Green Stain.
—— The inclusion of GelStar® and SYBR® Green Stains incesium chloride density gradient plasmid preparations
is not recommended. The effect of the dye on thebuoyant density of DNA is unknown.
—— These stains do not appear to interfere with enzymatic
reactions.
—— We recommend the addition of 0.1% to 0.3% SDS in the
prehybridization and hybridization solutions when
performing Southern blots on gels stained with thesedyes.
—— Double-stranded DNA-bound GelStar® or SYBR® Green
Stain fluoresces green under UV transillumination.
Gels that contain DNA with single-stranded regions
may fluoresce orange rather than green.
Decontamination
Staining solutions should be disposed of by passing
through activated charcoal followed by incineration of thecharcoal. For absorption on activated charcoal, consult
Sambrook, et al., pp. 6.16 - 6.19, (1989). Follow stateand local guidelines for decontamination and disposal of
Nucleic Acid staining solutions

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Section IV: Detection and Sizing of DNA in Agarose Gels
Detecting DNA with Ethidium Bromide

Introduction
Ethidium bromide is a fluorescent dye which detects
both single- and double-stranded DNA. However,
the affinity for single-stranded DNA is relatively low
compared to double-stranded DNA. Ethidium bromide
contains a planar group which intercalates between thebases of DNA and, when bound to DNA, results in an
increase in fluorescence yield. Ethidium bromidestained DNA is detected by ultraviolet radiation. At 254
nm, UV light is absorbed by the DNA and transmitted tothe dye; at 302 nm, and 366 nm, UV light is absorbedby the bound dye itself. In both cases, the energy isre-emitted at 590 nm in the red-orange region of thevisible spectrum.

■■ Materials–– Staining vessel larger than gel–– UV transilluminator–– Magnetic stir plate–– Magnetic stir bar■■ Reagents–– Ethidium bromide stock solution (10 mg/ml)–– 	1.0 g ethidium bromide–– 	100 ml distilled water–– 	Stir on magnetic stirrer for several hours–– 	Transfer the solution to a dark bottle–– 	Store at room temperature

Procedure–– Electrophoresis buffer or distilled water

For optimal resolution, sharpest bands and lowest
background, stain the gel with ethidium bromide following
electrophoresis.

Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

Ethidium bromide can also be included in the gel andelectrophoresis buffer (0.5 µg/ml) with only a minor lossof resolution. The electrophoretic mobility of DNA will be
reduced by approximately 15%.
Follow the steps below to stain DNA after electrophoresis
1.

Prepare enough working solution of ethidium bromide.
(0.5 - 1 µg/ml of ethidium bromide in distilled water orgel buffer) to cover the surface of the agarose gel.

Follow the steps below when including ethidium bromide
in the agarose gel
1.

Prepare agarose solution (see Section II).

2.

While the agarose solution is cooling, add ethidium
bromide to a final concentration of 0.1 to 0.5 µg/ml to
the solution.

Detection and Sizingof DNA in Agarose Gels

2.

Remove the gel from the electrophoresis chamber.

3.

Submerge the gel for 20 minutes in the ethidium bromide
solution.

3.

Gently swirl the solution.

4.

Remove the gel from the solution.

4.

Pour the gel into the casting tray.

5.

Submerge the gel for 20 minutes in a new container
filled with distilled water.

5.

Add ethidium bromide to the running buffer to a final
concentration of 0.5 µg/ml.

6.

Repeat in fresh distilled water.

6.

Load and run the gel (see Section III).

7.

Gels can be viewed with a hand-held or tabletop UV light.
For gel concentrations of 4% or greater, these times mayneed to be doubled. If after destaining the backgroundis still too high, continue to destain.

7.

Destain the gel by submerging the gel in distilled water for
20 minutes.

8.

Repeat in fresh distilled water.

9.

Gels can be viewed with a hand-held or tabletop UV lightduring or after electrophoresis.

Decontamination of ethidium bromide solutions
Decontamination of ethidium bromide solutions is
described in Sambrook, et al., pp. 6.16 - 6.17 (1989).
Follow local guidelines and regulations for ethidium
bromide decontamination and disposal.
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Section IV: Detection and Sizing of DNA in Agarose Gels
High Sensitivity Detection using The FlashGel® System

Introduction
The FlashGel® System uses a proprietary stain that is5-20 times more sensitive than ethidium bromide stain.
Samples prepared at DNA concentrations one-fifth of theconcentration typically required for ethidium bromide
stained gels will clearly resolve on FlashGel® Cassettes.
DNA levels of 5 ng per band or more are visible on the lighted
FlashGel® Dock under most ambient light conditions. DNA
levels as low as 0.1 ng per band can be detected on gel
images and photos. DNA levels can be adjusted to provide
best performance depending upon the image analysis
system used (Figure 1).

Because the system is so sensitive, a load volume of 5 μlor less is recommended for best performance. Samples
may be diluted with FlashGel® Loading Dye (for best
results), or with water or other common buffers (e.g.,
TE Buffer) before adding a loading dye. See the table in
Figure 2 for examples of sample and marker dilutions in
a FlashGel® Cassette compared to an ethidium bromide
stained agarose gel. The ethidium stained gel required atleast 4 times more sample than the FlashGel® Cassette.
One-tenth the concentration of the FlashGel® DNA Marker
and QuantLadder, and one-twentieth the concentrationof the PCR products were required for good sensitivity ofdetection on FlashGel® Cassettes compared to ethidium
bromide gel.

Figure1:
DNA concentration detectable with the FlashGel® System

Figure2:
FlashGel® Cassette vs. Ethidium Bromide Gel

1

2

3

4

5

6

7 8

FlashGel® Cassette

9 10 11 12 13
1

2 3

1

2

4 5

6

7

8

9 10 11 12 13­­— 2 kb­­— 500 bp

4 kb —
500 kb —ng/band75

50

25 12.5 6.25 3.13 1.56 0.78 0.39 0.20 0.10 0.05

1

2

3

4 5

6

7

8

100 kb —9 10 11 12 13

Ethidium Bromide Gel­­— 2 kb­­— 500 bp

3

4

5

6

7

8

9 10 11 12

4 kb —
500 kb —ng/band75

50

25 12.5 6.25 3.13 1.56 0.78 0.39 0.20 0.10 0.05

1.2% FlashGel® Cassettes, 12+1 well format. 275 volts for 7 minutes.
DNA concentrations are ng/band Lanes 1-12: Sample dilution series of
400 bp & 1,500 bp purified fragments (BioVentures) Lane 13: FlashGel®
DNA Marker. Photographed on UV Transilluminator or Dark Reader®
Transilluminator. Dark Reader® Transilluminator Gel illuminated on the
Dark Reader® with Dark Reader® orange filter in place. Photographedwith CCD imager (EtBr filter in place) at 2 second exposure. UVtransilluminator: Gel illuminated on UV transilluminator. Photographedwith CCD imager (EtBr filter in place) at 2 second exposure.

Lane Nos.Sample Type					
Lanes 1 & 7
FlashGel® DNA Marker
Lanes 2 & 8
FlashGel® QuantLadder
Lanes 3 & 9
Lonza 100 bp Ladder
Lanes 4 & 10
Lonza 50-2500 bp Marker
Lanes 5 & 11
285 bp b-Actin PCR
Lanes 6 & 12
294 bp Control PCR (Ambion)
Lane 13		
FlashGel® DNA Marker

100 kb —FlashGel® Loading Dye was added to all samples prior to loading.
Lanes 1&7: FlashGel® DNA Marker; Lanes 2&8: FlashGel® QuantLadder;
Lanes 3&9: Lonza 100 bp Ladder; Lanes 4&10: Lonza 50-2,500 bp
Marker; Lanes 5&11: 285 bp b-Actin PCR; Lanes 6&12: 294 bp Ambion
control PCR. Samples diluted with 1X FlashGel® Loading Dye prior toloading. Dilutions and load volumes optimized for each sample in eachgel system. See table below.

Load volumes and dilutions
FlashGel® Cassette		
5 µl 1:5 dilution		
5 µl 1:5 dilution		
3 µl 1:15 dilution		
3 µl 1:5 dilution		
5 µl 1:50 dilution		
5 µl 1:50 dilution		
5 µl 1:5 dilution

Ethidium Bromide Gel
10 µl undiluted
10 µl undiluted
12 µl 1:15 dilution
12 µl 1:5 dilution
2 µl undiluted PCR rxn
2 µl undiluted PCR rxn

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Section IV: Detection and Sizing of DNA in Agarose Gels
High Sensitivity Detection using The FlashGel® System ­— continued

Refer to The FlashGel® System Protocol for complete user
instruction, safety and environmental precautions.
Some components and technology of the FlashGel® System are sold underlicensing agreements. The nucleic acid stain in this product is manufactured
and sold under license from Molecular Probes, Inc., and the FlashGel®
Cassette is sold under license from Invitrogen IP Holdings, Inc, and is foruse only in research applications or quality control, and is covered bypending and issued patents. The FlashGel® Dock technology contains
Clare Chemical Research, Inc. Dark Reader® Transilluminator technology
and is covered under US Patents 6,198,107; 6,512,236; and 6,914,250.
The electrophoresis technology is licensed from Temple University and iscovered under US Patent 6,905,585.

Detection and Sizingof DNA in Agarose Gels

References
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