Loading and Running
DNA in Agarose Gels

Section III: Loading and Running DNA in Agarose Gels

In This Section
DNA Loading
Loading Buffers
Optimal Voltage and Electrophoretic Times
Fast Running Protocols for High Resolution
in MetaPhor® Agarose Gels
References

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Section III: Loading and Running DNA in Agarose Gels
DNA Loading

Introduction
The amount of DNA to load per well is variable. Most
important are the quantities of DNA in the bands ofinterest.
Optimal DNA loading amountThe amount of DNA that may be loaded on a gel depends
on several factors:—— Well volume—— Fragment size: The capacity of the gel drops sharply
as the fragment size increases, especially over a fewkilobases—— Distribution of fragment sizes—— Voltage gradient: Higher voltage gradients are better
suited to DNA fragments <1 kb, lower voltages are
better suited to fragments >1 kb—— Detection Method: The least amount of dsDNA in asingle band that can be reliably detected with ethidium
bromide is approximately 10 ng, with GelStar® Nucleic
Acid Gel Stain is approximately 20 pg and with SYBR®
Green I Stain is 60 pg

The optimal amount of DNA to load in the well may be
calculated by the fraction of the total DNA which is in theband of interest, represented by the following:
Fragment of interest (kbp)
X 100 = % DNA in
Size of DNA sample (kbp)band of interest
Note: The most DNA compatible with a clean sharpband is approximately 100 ng.
For example:
The size of your DNA sample is 48.5 kbp and when run
on the gel 8 fragments are separated. Your fragment ofinterest is 2.3 kbp.
Calculation:
2.3 kbp48.5 kbp X 100 = 4.7% DNA in fragment ofinterest
If you load 1 µg of DNA, then 4.7% of the 1 µg of loaded
sample will appear in your fragment of interest (47 ng).

Overloaded DNA results in trailing and smearing, aproblem that will become more severe as the size of theDNA increases.

Loading and Running
DNA in Agarose Gels

The photograph below shows the effect of overloaded
and underloaded DNA on an agarose gel. Where samples
are loaded in excess (0.5 µg/lane), you can see bandbroadening and smearing of the larger molecular weightfragments. Where samples are underloaded, you lose thesmall molecular weight fragments. The optimum loadinglevel for the marker used in the photograph is 0.1 µg/lane.
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4

5– 23,130 bp										– 6,557 bp									– 2,322 bp							– 564 bp

Separation of DNA markers in a 1% SeaKem® GTG® Agarose gel
prepared and run in 1X TBE Buffer. Hind III digested lambda DNA(Boehringer Mannheim) was loaded from left to right at 0.025, 0.05,0.1, 0.2 and 0.5 µg/lane. 20 cm long gels were run at 6 V/cm for 2hours, 5 minutes.

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Section III: Loading and Running DNA in Agarose Gels
Loading Buffers

IntroductionFicoll® based loading buffers

Gel loading buffers serve three purposes in DNA
electrophoresis:

To increase the sharpness of DNA bands, use Ficoll® (Type400) Polymer as a sinking agent instead of glycerol. The
use of the lower molecular weight glycerol in the loading
buffer allows DNA to stream up the sides of the well before
electrophoresis has begun and can result in a U-shapedband. In TBE gels, glycerol also interacts with borate which
can alter the local pH.

—— Increase the density of the sample: This ensures thatthe DNA will drop evenly into the well—— Add color to the sample: Simplifies loading—— Add mobility dyes: The dyes migrate in an electric field
towards the anode at predictable rates. This enables
one to monitor the electrophoretic process
Loading buffers
At least five loading buffers are commonly used for agarose
gel electro­phoresis. They are prepared as six-foldconcentrated solutions. If needed, 10X solutions of each buffer
can also be prepared. Alkaline loading buffer is used when
performing alkaline gel electrophoresis.

Loading Buffer

6X Recipe

4°C

GlycerolBased

30% Glycerol in distilled water
0.25% Bromophenol Blue
0.25% Xylene cyanol FF

4°C

Ficoll®
Based

15% Ficoll® (Type 400)
Polymer in distilled water
0.25% Bromophenol Blue
0.25% Xylene cyanol FF

Alkaline

Loading buffer that is too high in ionic strength causes
bands to be fuzzy and migrate through the gel at
unpredictable rates. Ideally, the DNA sample should beresuspended in the same solution as the running buffer. If
this is not possible, use a sample buffer with a lower ionicstrength than the running buffer.
The photograph below shows the effect of high salt
concentrations in loading buffers on DNA resolution.

Storage Temperature

40% (w/v) Sucrose
0.25% Bromophenol Blue
0.25% Xylene cyanol FF

SucroseBasedSample preparation

1room
temperature

300 mN NaOH
4°C
6 mM EDTA
18% Ficoll® (Type 400) Polymer
in distilled water
0.15% Bromocresol Green
0.25% Xylene cyanol FF

Lonza offers ready-to-use DNA loading buffers. Refer topage 41 for information.

234 567 8 9Loading and Running
DNA in Agarose Gels

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– 23 kb– 9.4 kb

– 4.3 kb
– 2.33 kb– 0.56 kb

Separation of Hind III digested lambda DNA (Invitrogen, Inc.) marker(5 µg/lane) in a 1% SeaKem® GTG® Agarose gel prepared and run in1X TAE Buffer. 20 cm long gels were run at 6 V/cm for 2 hours. Thesample buffer was mixed with varying amounts of NaCl to obtain
different final salt concentrations.
Lane 1: 4 M NaCl, Lane 2: 3 M NaCl,
Lane 3: 1 M NaCl, Lane 4: 0.5 M NaCl,
Lane 5: 0.25 M NaCl and Lanes 7 - 9: No addition of NaCl.

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Section III: Loading and Running DNA in Agarose Gel
Optimal Voltage and Electrophoetic Times

Optimal voltage

Voltage table

The distance used to determine voltage gradients is thedistance between the electrodes, not the gel length.
If the voltage is too high, band streaking, especiallyfor DNA ≥12 kb - 15 kb, may result. When the voltage istoo low, the mobility of small (≤1 kb) DNA is reducedand band broadening will occur due to dispersion
and diffusion.

The table below provides a quick reference for optimal
voltage for DNA electrophoresis.

The photographs below show the effect of voltage on
small DNA. The small fragments on the gel run at 1 V/cm
show severe band broadening and fuzziness.
The gel run under 5 V/cm has sharp bands both in thesmall fragments and the larger fragments. Buffer also
plays a role in band sharpness.
The photographs below show the effect of voltage andbuffer on large DNA. When large DNA is subjected to very
high voltage, smearing occurs.

Recommended Voltages and Buffers Related to DNA Sizeand ApplicationSize≤1 kb
1 kb to 12 kb>12 kb

Voltage
5 V/cm
4 - 10 V/cm
1 - 2 V/cm

Buffer				
Recovery		 Analytical
TAE
TBE
TAE
TAE/TBE
TAE
TAE

note: MetaPhor® Agarose can also be run at very highvoltages to achieve 1% - 2% resolution. See “Fast Running
Protocols for High Resolution in MetaPhor® Agarose Gels,”
this chapter.
Optimal electrophoretic time
The gel should be run until the band of interest has
migrated 40% - 60% down the length of the gel (see the Dye
Mobility Table). Band broadening resulting from dispersion
and diffusion results in a decrease in resolution in thelower third of the gel. Resolution may also be decreased
in smaller gels, since longer electrophoretic runs result ingreater separation between two fragments.

Gel 1
AB

Gel 2
ABLoading and Running
DNA in Agarose Gels

1 2Gel 1

3 456

1 2Gel 2345 6

1,353 kb –– 622 kb

310 kb –194 kb –– 160 kb– 110 kb
– 67 kb

Separation of DNA markers in 3% NuSieve® 3:1 Agarose Gels. Lane A: Hae III
digested φX174 DNA (New England Biolabs); 0.5 µg/lane. Lane B: Msp I
digested pBR322 DNA (New England Biolabs);
0.5 µg/lane. Gel 1: 21 cm long gel run at 1 V/cmin 1X TAE Buffer (recirculating) for 16 hours.
Gel 2: 20 cm long gel run at 5 V/cm in 1X TBE Buffer for 2 hours, 10minutes.

Separation of Gensura’s 5 kb and 2 kb DNA ladders in 0.5% SeaKem® LEAgarose gels. Odd numbered lanes are 5 kb ladders; even numbered lanes
are 2 kb ladders. Lane 1: 5 ng/band;
Lane 2: 5 ng/band; Lane 3: 20 ng/band;
Lane 4: 20 ng/band; Lane 5: 60 ng/band;
Lane 6: 60 ng/band. Gel 1: 20 cm long gel run
at 8 V/cm in 1X TBE Buffer without recirculation.
Gel 2: 21 cm long gel run at 1 V/cm in 1X TAE Buffer with recirculation.

The FlashGel® System (pages 18-25) enables very fast
(5 minute) high voltage separation of DNA fragments 10
bp to 10 kb.

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Section III: Loading and Running DNA in Agarose GelsFast Running Protocols for High Resolution in MetaPhor® Agarose Gels

Introduction■■ Materials–– Horizontal electrophoresis chamber to accommodatea 20 cm long gel or vertical electrophoresis chamber

The protocols in the following section describe how
to increase resolution to a 1% size difference with
DNA between 100 bp and 500 bp, and decrease yourelectrophoretic time to 1.5 hours.
A standard horizontal submarine gel apparatus canbe used to achieve resolution which is comparable topolyacrylamide gels at ≤8%. MetaPhor® Agarose can also
be used to achieve similar resolution in a standard verticalgel electrophoretic system.

–– Power supply–– Recirculator-chiller water bath■■ Reagents–– Electrophoresis buffer (TAE or TBE)–– MetaPhor® Agarose–– GelStar® or SYBR® Green Nucleic
Acid Gel Stains or ethidium bromide solution

The photographs below show the resolution achieved in 4%MetaPhor® Agarose gels, using the same markers in threedifferent formats.
Electrophoretic conditions
VerticalFormat
Resolution
1% (≤500 bp)
Run Time
1 - 1.5 hours
Gel Concentration 3% - 4%
Gel Length
16 cm - 26 cm
Gel Thickness
1 mmGel Buffer
1X TAE or 1X TBE
Running Buffer 1X TAE or 1X TBE
TBEVoltage*
17 V/cm
Temperature
ambient

Rapid Horizontal		
Format1% (≤500 bp)
1.5 hours
3% - 4%20 cm
3 mm
1X TAE or 1X TBE
0.5X TAE or 0.5X TBE
17 V/cm
15°C***V/cm is determined by the total voltage divided by the interelectrodedistance in cm.
**Circulate electrophoresis buffer with a recirculator-chiller water bath.

StandardHorizontal
Format1 2 3 4 5 6

VerticalFormat

12 3 4 5 6

RapidHorizontal
FormatCaution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.
Fast running protocol for horizontal gels
NOTE: This protocol cannot be used with only a peristaltic

pump in the cold room; the gel will melt.
1.

Prepare a 3 - 4% MetaPhor® Agarose gel in 1X
electrophoresis buffer.

2.

Cast a 3 mm thick, 20 cm long agarose gel.

3.

Allow the gel to solidify at room temperature.

4.

Place at 4°C for 30 minutes.

5.

Place the gel in the electrophoresis chamber.

6.

Use 0.5X TBE or 1X TAE Running Buffer in theelectrophoresis chamber.

7.

Add the running buffer to a depth of 3 mm over the surfaceof the gel.

8.

Load 20 ng - 50 ng of DNA. If this cannot be
estimated, load varying amounts of the sample.
The sharpest bands are seen with small(5 µl - 10 µl) loading volumes.

9.

Run the gel at 17 V/cm (interelectrode distance).

10.

When the sample has left the well and moved into thegel, begin recirculating the electrophoresis buffer.

11.

Chill and circulate the electrophoresis buffer with arecirculator-chiller water bath.

12.

Run the gel for approximately 1.5 hours.

1 2 3 4 5 6– 622 bp– 527 bp

– 160 bp
– 147 bp
– 76 bp– 34 bp

4 hours

1.5 hours

Loading and Running
DNA in Agarose Gels

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1.5 hours

Lane 1: 4 bp linker ladder; Lane 2: 8 bp linker ladder; Lane 3: 12 bp
linker ladder; Lane 4: 600 ng of 100 bp ladder; Lane 5: 400 ng of
pBR322/Msp I digest + 600 ng of 100 bp ladder; Lane 6: 400 ng of
pBR322/Msp I digest.

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Section III: Loading and Running DNA in Agarose Gels
Fast Running Protocols for High Resolution in MetaPhor® Agarose Gels — continued

Troubleshooting
The fast running protocol will not work if the buffer is
not chilled or recirculated. The photograph to the rightshows the effect of DNA resolution on running the gel in acold room (4°C), without buffer recirculation.

– 622 bp

Fast running protocol for vertical gels
1. 	Cast a vertical agarose gel in 1X electrophoresis
buffer following the Vertical Gel Casting Instructions onpage 86.

2.

Carefully flush the wells with running buffer.

3.

Load 20 ng - 50 ng of DNA in the band of interest. If
this cannot be estimated, load varying amounts ofthe sample. The sharpest bands are seen with a small(5 µl - 10 µl) loading volume.

4.

Run the gel at 17 V/cm interelectrode distance.

5.

Run the gel approximately 1 - 1.5 hours.

– 100 bp

Msp I digest of pBR322 (New England Biolabs),
400 ng/lane and Lonza's 100 bp DNA Ladder,
600 ng/lane run on a 4% MetaPhor® Agarose gel prepared in 1X TAEBuffer. The gel was run at 17 V/cm in 0.5X TAE Buffer for 90 minutes in
the cold room without buffer recirculation.

Note: The gel melted and was allowed to resolidify prior tostaining.

Loading and Running
DNA in Agarose Gels
References
FMC BioProducts, Resolutions 9(1), 1992.
Rickwood, et al., Nucleic Acid Electrophoresis: A Practical Approach, 2nd edition,
Oxford University Press, 1990.
Sambrook J., et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring
Harbor Laboratory Press, 1989.
Vandenplas S., et al., J. Medical Genetics 21: 164 - 172, 1984.

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