FAQs

Section I: Frequently Asked Questions

In This Section
Nucleic Acid FAQs
Protein Analysis FAQs

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Section I: Frequently Asked Questions
Nucleic Acid FAQs

Electrophoresis and Agarose

Nucleic Acid FAQs

Q.	What buffer conditions give me best resolution
for agarose electrophoresis?
A.
For small DNA fragments (<1,000 bp) whenrecovery is not necessary, we recommend theuse of 1X TBE Buffer. For any given concentrationof agarose, gels made with TBE Buffer give sharperbands than gels made with TAE Buffer. TBE results
in better resolution for closely spaced DNA bands.
For large DNA fragments (>15,000 bp), 1X TAE
Buffer enhances separation of large DNA. Since
TAE has a lower buffering capacity, it may benecessary either to recirculate the buffer, or
periodically mix the buffer between the anodal
and cathodal chambers when electrophoresing
for an extended period of time. The time to buffer
depletion can vary with the volts/hour and thesize of chamber used.
Whichever buffer you use, the depth over the gelshould be 3 to 5 mm deep. Less buffer, and you
risk the chance of the gel drying out. Excessive
buffer will decrease the resistance of the circuit
between the anode and cathode, which results
in a decreased voltage gradient through the gel.
This causes inefficient DNA mobility, excessive
heating, and band distortion.
Q.
A.

How should I cast my gels to get the best
resolution?
The first concern is the thickness of the gel. We
usually cast gels 3 mm to 4 mm thick. The gelvolume needed can easily be estimated bymeasuring the surface area of the casting chamber,then multiplying by the gel thickness. Thinner gelscan be cast on GelBond® Support Film, and/or castin a vertical apparatus.
The thickness of the comb in the direction of
the electrical field can also profoundly affectthe resolution. A thin comb (1 mm) will result in
sharper DNA bands. With too thick a comb, theseparated DNA bands will be quite broad.

Q.
A.

Q.
A.

My DNA bands are sometimes wavy, but usually
only in one or two lanes. What causes this?
Dried agarose on the comb teeth is a frequentcause of this problem. Prior to casting your gel,check the comb teeth for residual dried agarose.
If not removed, this will attach to the newly cast
agarose and fracture the well upon comb removal.
This is usually not observable until the gel is on
the transilluminator. Additionally, care must be
taken during comb removal, particularly with low
melting temperature agaroses. Well integrity may
be maintained in these agaroses by pre-chilling
the gel to 4°C for 30 minutes and/or by flooding
the gel with cold buffer prior to removing thecomb.
How much DNA should I load per well?
The amount to load per well is variable. What ismost important is how much DNA there is in the
bands you wish to resolve. The least amountof DNA that can be consistently detected withethidium bromide is about 10 ng. The most DNA
you can have in a band and still get a sharp, clean
band on an ethidium bromide stained gel is about
100 ng. These amounts will be less on gels stainedwith more sensitive stains such as GelStar® Stain.
On a GelStar® Stained Gel it is possible to detect as
little as 20 pg dsDNA.
The optimal amount of DNA to load in the well is
calculated by the fraction of the total DNA which isin the band of interest. If you are unsure of how
much DNA is present, load varying amounts inseveral lanes if possible.
To further increase the sharpness of the bands,
use a Ficoll® based loading buffer such as Lonza
DNA Loading Buffer (Cat. No. 50655) instead of
sucrose-based or glycerol-based loading buffers.
The use of lower molecular weight glycerol willallow DNA to stream up the sides of the well before
electrophoresis which results in U-shaped bands.
Loading buffer that is too high in ionic strength
can cause the bands to be fuzzy. In the ideal
situation, the DNA sample should be suspended
in the same solution as the running buffer. If thisis not possible, use a sample buffer with a lower
ionic strength than the running buffer.

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Nucleic Acid FAQs — continued

Electrophoresis and Agarose

Q.

Q.
A.

A.

Q.
A.

Q.

A.

At what voltage should I run an agarose gel?
We recommend running agarose gels at 4 – 10
volts/cm (cm is determined by measuring theinterelectrode distance, not the gel length) undernormal horizontal electro­phoretic conditions. If thevoltage is too high, band streaking, especially for
DNA >15 kb, may result. When the voltage is too low,
the mobility of small (<1,000 bp) DNA is reduced
and band broadening will occur due to diffusion.
MetaPhor® Agarose gels separate DNA optimallyat 4.5-5 volts /cm in standard horizontal electro­
phoresis systems. Higher voltages result in adecrease in the resolution of DNA separation,
mainly due to gel overheating.
Another special case is the separation of large (>15kb) DNA fragments using conventional horizontal
electro­phoresis. The best separations in thisinstance are obtained at a voltage gradient of <5
volts /cm.
What is the difference between NuSieve® 3:1 and
NuSieve® GTG® Agaroses?
NuSieve® 3:1 Agarose is a standard melting
temperature agarose. The resolution range for
NuSieve® 3:1 Agarose is 50 bp – 1000 bp.
NuSieve® 3:1 Agarose is designed for analyticalelectrophoresis; its high gel strength also makes it
ideal for use in various blotting techniques.
NuSieve® GTG® Agarose is a low melting
temperature agarose (≤65°C at 4%). The
resolution range for this agarose is 50 bp – 1000
bp. NuSieve® GTG® Agarose is recommended forin-gel applications such as cloning or ligation andtransformation.
GTG® stands for Genetic Technology Grade™. GTG®
Grade Agarose is recommended for preparative
DNA electrophoresis, or when further enzymatic
manipulation of DNA is required. These agaroses
are extensively tested to ensure maximum
compatibility with standard molecular biology
techniques.
What is the difference between SeaKem® LE and
SeaKem® GTG® Agaroses?
SeaKem® LE and SeaKem® GTG® Agaroses areboth standard melting temperature agaroses. The
resolution range for these agaroses is 100 bp to23,000 bp.
SeaKem® LE Agarose is ideal for routine analysis
of DNA. SeaKem® GTG® Agarose is a Genetic
Technology Grade™ Agarose, specifically designed
for preparative DNA electrophoresis.

What is the difference between SeaPlaque® and
SeaPlaque® GTG® Agaroses?
SeaPlaque® and SeaPlaque® GTG® Agaroses areboth low melting temperature agaroses (≤65°C at1.5%). The resolution range for these agaroses is
200 bp – 25,000 bp.
SeaPlaque® Agarose is recommended forpre­parative DNA electrophoresis. SeaPlaque® GTG®
Agarose is a Genetic Technology Grade™ Agarose,
recommended for direct enzymatic manipulationof nucleic acids in remelted agarose (in-gel
reactions). It is also compatible with PCR andsequencing reactions carried out in the presence ofthe remelted gel.

Nucleic Acid FAQs

Returnto MainPrecast Agarose Gels
Q.
A.

Q.
A.

Q.
A.

Do I need to purchase a special chamber to use
Reliant® and Latitude® Precast Gels?
Reliant® and Latitude® Precast Agarose Gels
are designed to run in standard horizontal
electrophoresis chambers. As long as there isroom on the chamber platform for the gel, thechamber should be suitable. Measure the chamber
platform and check against precast gel size to be
sure. For example, the OWL® Centipede™ Chamber isideal with the 14 cm ✕ 24 cm Latitude® HT Precast
Gels; the OWL® B1 EasyCast™ Chamber is good with
Reliant® Precast Agarose Gels; and the Latitude®
Chamber is perfect with Latitude® Midigels. Resultswith different chambers will vary depending on
differences in chamber size and construction.
Can I use the FlashGel® System for both DNA andRNA?
Yes. We offer FlashGel® Cassettes and Reagentsfor both DNA and RNA. The FlashGel® Dock may be
used for both cassette types as there is no risk ofcontamination.
Are the materials in the FlashGel® Cassettehazardous?
The stain in the FlashGel® Cassette is present atsuch low levels that it is not considered hazardous
according to OSHA and EU hazard criteria. A copy of
the MSDS is available online. The stain in the cassette isa potential mutagen. Wear gloves, safety glasses anda lab coat when handling. Use the same precautions
when handling and disposing of the cassettes as you
would ethidium bromide stained gels.

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Section I: Frequently Asked Questions
Protein Analysis FAQs

PAGEr® Precast Gels

Protein Electrophoresis

Q.

Which PAGEr® Precast Gels will fit my gel
chamber?

Q.

How do I make the transfer, running, and sample
buffers?

A.

PAGEr® Precast Gels are available in 9 cm × 10 cm
and 10 cm × 10 cm sizes and fit most standard
mini-vertical systems. Some chambers may
require modifications for optimal fit with PAGEr®
Precast Gels.

A.

Tris-Glycine Gels (Tris-HCl Buffer System)

Protein Analysis
FAQs

Standard Vertical Systems
PAGEr® Minigel Chamber

PAGEr® Gels
9 cm × 10 cm
10 cm × 10 cm gels

Bio-Rad® MiniPROTEAN® II, MiniPROTEAN® 3 orReady Gel® Cell Systems

9 cm × 10 cm gels

TowbinTransfer Buffer (1X)

Running Buffer (1X)

Sample Buffer (1X)

0.025 M Tris base25 mM Tris Base

62.5 mM Tris-HCl, pH
6.8

0.192 M Glycine

192 mM Glycine

2% SDS*

0.05 – 0.1% SDS*

0.1% SDS*

10% Glycerol

20% Methanol

Reverse the inner core gasket so the flat side faces outward.

Novex® XCell SureLock® Mini-Cell

2.5% bME(2-mercaptoethanol)*

9 cm × 10 cm
10 cm × 10 cm gels

Request the spacer for the XCell SureLock®, Mini-Cell Chamber from
Scientific Support, (Cat. No. 59900).

FisherBiotech® Vertical Minigel FBVE121,
Owl Separations Systems Wolverine™ P82

10 cm × 10 cm gels

Chamber comes with 2 sets of wedges. Use the thinner wedges for the PAGEr® Gold Gels.

FisherBiotech® Vertical Minigel FB-VE101,
Owl Separations Systems Penguin™ Model P8DS

10 cm × 10 cm gels

Hoefer® Mighty Small (SE250)

9 cm × 10 cm
10 cm × 10 cm gels

*Omit for native proteins
For best results use Lonza AccuGENE® Electrophoresis Buffers

Q.

What is the difference between gradient vs. homo­
geneous (single concentration) gels? Which one
should I use?

A.

Gradient gels are suitable for a wide range of size
resolutions. A homogeneous or single concen­
tration gel is appropriate where the proteins ofinterest are known to be within a narrow size
range.
How much protein should I load on the gel?

Request adaptor for these chambers from Scientific Support, (Cat. No. 59902).

Replace the buffer chamber with a ‘Deep lower buffer chamber for the SE260’,
order number 80-6148-78, from GE Healthcare.

Daiichi 2, ISS chambers

10 cm × 10 cm gels

To run one gel: Place one 10 × 10 cm cassette on wedge side of chamber. Use suitable buffer damon the other side. Use regular Daiichi/ISS wedges.
To run two gels: Widen the hole on the yellow port of the inner core. Replace the long arm wedges
with modified wedges.This chamber modification and new wedges are available from ScientificSupport.

Novex® XCell II

9 cm × 10 cm or 10 cm × 10 cm gels

Hoefer® Mighty Small (SE260)

9 cm × 10 cm or 10 cm × 10 cm gels

EC 120 Mini Vertical Gel System

9 cm × 10 cm or 10 cm × 10 cm gels

Biometra® Mini V Chamber

9 cm × 10 cm gels

CBS Scientific MGV System,(10 cm × 8 cm units)

9 cm × 10 cm gels

Sigma-Aldrich Mini Techware(11.3 cm × 10 cm units)

10 cm × 10 cm gels

Zaxis System 2000

10 cm × 10 cm gels

Hoefer® Mini VE

10 cm × 10 cm gelsQ:

Do Lonza PAGEr® Precast Gels contain a stacking
gel? What is the purpose of the stacking gel?

A.

PAGEr® Gold Precast Gels contain a 4% stacking gel,pH 8.6. The purpose of this stacking gel is to allow
the proteins to accumulate and condense (i.e.
stack) at the stacking/resolving gel boundary.
This stacking effect results in superior resolution
within the running gel.
I would like to run a native or nondenaturing gel.
What can I use?

Q.
A.

0.01% Bromophenol
Blue

Q.
A.

Protein load levels will vary depending uponsample purity and staining method used. For
highly purified proteins, 0.5 μg to 5 μg protein perlane on a minigel is generally sufficient. Complete
mixtures such as cell lysates may require as muchas 50 μg protein per lane.

Protein stain detection limits
Protein Stain
Coomassie® Blue Stain
Silver Stain
SYPRO® Red Protein Gel Stain
SYPRO® Ruby Protein Gel Stain
SYPRO® Tangerine Protein Gel Stain

Lower Detection Limit(Protein / Band)
30 ng
2 ng
4 ng – 8 ng
2 ng – 8 ng
4 ng – 8 ng

NOTE: Limits are based on optimal detection methods for each stain.

PAGEr® Precast Gels do not contain SDS or any
other denaturing agents (e.g. DTT and b-ME).
Additionally, you would use a Tris-Glycine
Running Buffer that does not contain SDS.

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Protein Analysis FAQs — continued

Q.

What is the best membrane to use for Western
blotting?

A.

Use this table to find a suitable membrane.

Nitrocellulose
Hydrophobic binding

PVDF
Hydrophobic binding

Nylon
Hydrophobic
and electrostatic
binding

General purpose
membrane

SDS tolerant

Stable if baked

Low backgroundLow strength
Becomes brittle if baked

High background
High strength
Suitable for protein
sequencing

High background
High strength
Least suitable forWestern transfer

Q.

What are the benefits of using agarose for protein
gel electrophoresis?

A.

Protein electrophoresis in agarose gels is analternative approach to using polyacrylamidegels and provides several benefits:–– Separate high molecular weight proteins( > 600 kDa)

ProSieve® Protein Markers
Q.

In what applications do you recommend using the
ProSieve® Color Protein Marker vs. the ProSieve®
Protein Marker?

A.

The ProSieve® Color Protein Markers are ideal
for monitoring protein separation during
electrophoresis and confirming protein transfer inWestern blotting. They are not recommended for
precise sizing of protein samples in SDS-PAGE.
The ProSieve® Protein Markers are recommended
for the most accurate sizing of protein samples
in SDS-PAGE. These markers contain 10 proteins
with exact masses of 5 kDa, 10 kDa, 15 kDa,
25 kDa, 35 kDa, 50 kDa, 75 kDa, 100 kDa, 150 kDa,
and 225 kDa.

Protein Analysis
FAQs

Returnto Main–– Easy to prepare and handle–– Efficient recovery of proteins–– Excised proteins can be used to immunize
animals directly for antibody production–– Non-toxic–– Run gels using either a vertical or horizontal
apparatus

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