9 DNA Electrophoresis2010

All Fermentas products are
manufactured in class D clean room

facilities, qualified and certified as
per EU directives and ISPE guidelines.
Fermentas quality assurance is carried
out according to ISO9001 quality and
ISO14001 environmental management
systems, guaranteeing batch-to-batch
reproducibility. Integration of our clean
room and ISO systems ensures stability
and the absence of contaminants in all
of our products.
DNA ELECTROPHORESIS
Selection Guide................................................................................................................408
Products............................................................................................................................. 413
NoLimits Individual DNA Fragments and Custom DNA Ladders............................. 413

GeneRuler and OGeneRuler DNA Ladders (10-20000bp) . ................................... 414
GeneRuler 1kb DNA Ladder..................................................................................... 415
GeneRuler 1kb Plus DNA Ladder.............................................................................. 415
GeneRuler DNA Ladder Mix...................................................................................... 415
GeneRuler 100bp DNA Ladder................................................................................. 415
GeneRuler 100bp Plus DNA Ladder......................................................................... 415
GeneRuler 50bp DNA Ladder................................................................................... 415
GeneRuler Ultra Low Range DNA Ladder................................................................... 415
GeneRuler Low Range DNA Ladder........................................................................... 415
GeneRuler High Range DNA Ladder.......................................................................... 415
GeneRuler Express DNA Ladder................................................................................ 415
MassRuler DNA Ladders, readytouse (80-10000bp) ........................................... 418
MassRuler Low Range DNA Ladder........................................................................... 418
MassRuler Express LR Forward DNA Ladder............................................................. 418
MassRuler Express LR Reverse DNA Ladder............................................................. 418
MassRuler High Range DNA Ladder.......................................................................... 418
MassRuler Express HR Forward DNA Ladder............................................................. 418
MassRuler Express HR Reverse DNA Ladder............................................................. 418
MassRuler DNA Ladder Mix...................................................................................... 418
MassRuler Express Forward DNA Ladder Mix............................................................ 418
MassRuler Express Reverse DNA Ladder Mix............................................................ 418
FastRuler DNA Ladders, readytouse (10-10000bp) . ............................................ 420
FastRuler Ultra Low Range DNA Ladder.................................................................... 420
FastRuler Low Range DNA Ladder............................................................................ 420
FastRuler Middle Range DNA Ladder........................................................................ 420
FastRuler High Range DNA Ladder............................................................................ 420
ZipRuler Express DNA Ladder Set, readytouse (100-20000bp) .......................... 421
ORangeRuler DNA Ladders, readytouse (10-6000bp) ........................................ 422
ORangeRuler 5bp DNA Ladder................................................................................ 422
ORangeRuler 10bp DNA Ladder.............................................................................. 422
ORangeRuler 100bp DNA Ladder............................................................................ 422
ORangeRuler 200bp DNA Ladder........................................................................... 422
ORangeRuler 500bp DNA Ladder........................................................................... 422
ORangeRuler 100+500bp DNA Ladder................................................................... 422
Conventional Lambda DNA Markers (15-48502bp) . ................................................. 424
Lambda DNA/EcoRI Marker, 1.................................................................................... 424
Lambda DNA/HindIII Marker, 2................................................................................... 424
Lambda DNA/EcoRI+HindIII Marker, 3........................................................................ 424
Lambda pUC MIx Marker, 4..................................................................................... 424
Lambda DNA/Eco47I (AvaII) Marker, 13...................................................................... 424
Lambda DNA/Eco91I (BstEII) Marker, 15..................................................................... 424
Lambda DNA/Eco130I (StyI) Marker, 16...................................................................... 424
Lambda Mix Marker, 19.............................................................................................. 424
Lambda DNA/PstI Marker, 24..................................................................................... 424
Conventional Phage and Plasmid DNA Markers (8-1353bp) .................................... 426
pBR322 DNA/BsuRI (HaeIII) Marker, 5........................................................................ 426
pUC Mix Marker, 8...................................................................................................... 426
FX174 DNA /BsuRI (HaeIII) Marker, 9......................................................................... 426
FX174 DNA /HinfI Marker, 10..................................................................................... 426
pBR322 DNA/AluI Marker, 20..................................................................................... 426
pUC19 DNA/MspI (HpaII) Marker, 23.......................................................................... 426
Loading Dyes................................................................................................................. 428
TopVision Agarose......................................................................................................430
Electrophoresis Buffers...............................................................................................430
Protocols and Recommendations................................................................................ 431
9.1. General recommendations for DNA electrophoresis..................................................... 431
9.2. Preparation of agarose gels for DNA electrophoresis................................................... 431
9.3. Preparation of gels for PAGE...................................................................................... 432
9.4. Preparation of DNA samples for electrophoresis.........................................................433
9.5. Preparation of DNA ladders/markers for electrophoresis.............................................433
9.6. Labeling of DNA ladders/markers..............................................................................433
9.7. Separation of Express DNA ladders in different electrophoresis conditions...................434
Troubleshooting Guide....................................................................................................435
Selection Guide
PProduct lines of DNA ladders and markers
Features
9. DNA ELECTROPHORESIS
DNA ladder/marker
group
Separation
Range,bp time on
agarose
Main feature
NoLimits Custom
10-20000
DNA Ladders
Formulated
according
Composition
to customer
dependent
specifications.
Bulk orders
GeneRuler and
OGeneRuler
DNA Ladders
10-48502
10min1.5 h
MassRuler
DNA Ladders
80-10000
10-45min
FastRuler
DNA Ladders
10-10000
8-14min
ORangeRuler
DNA Ladders
10-6000
45min1.5 h
ZipRuler Express
100-20000 10-20min
DNA Ladders
Conventional
15-48502
Lambda DNA
Markers
Conventional
Phage and Plasmid 8-1353
DNA Markers
Note
Formulation
Mixture of
individual,
chromatography-purified
DNA fragments
Mixture of
individual,
Accurate DNA
chromatoquantification
graphy-purified
DNA fragments
Mixture of
5 individual,
Fast separation.
chromatoin short run
graphy-purified
DNA fragments
Ligation
Step ladders of products of
5, 10, 20, 50,
10, 15, 20,
100, 200, 500bp 50, 100, 200,
increments
500bp DNA
fragments
Mixture of
Fast and accurate
individual,
sizing of broad
chromatorange DNA
graphy-purified
fragments
DNA fragments
Applications
Quanti Labeling* with
fication
Fast
T4 PNK Klenow
Electrophoresis
Page
Agarose
PAGE
413
6X DNA
Loading
In TE buffer or .
Dye or .
readytouse
6X Orange
Loading Dye
414
6X
Readytouse MassRuler
Loading Dye
418
6X
MassRuler
Readytouse Loading Dye, .
6X Orange
Loading Dye
420
Individual,
Any
chromatoIn TE buffer or . Fermentas
graphy-purified readytouse Loading Dye
DNA fragments
upon request
Sizing of broad
range DNA
fragments
Readytouse
6X Orange
Loading Dye
422
Readytouse
6X Orange
Loading Dye
421
In TE buffer or . 6X DNA
readytouse Loading Dye
424
Digested Phage
In TE buffer or . 6X DNA
Classical markers and plasmid
readytouse Loading Dye
DNA
426
45min-18 h Classical markers
45min1.5 h
Origin
Loading
dyes
supplied
Digested
Lambda DNA
Not all the DNA ladders/markers of the group are suitable for the indicated application.
* Only DNA ladders/markers supplied in TE buffer can be labeled.
www.fermentas.com
408
For patent and license information see p.557
Range selection for DNA Ladders and Markers
10bp
100bp
1kb
10kb
10-300bp
25-700bp
50-1000bp
75-20000bp
100-1000bp
100-3000bp
100-5000bp
100-10000bp
250-10000bp
10171-48502bp
50kb
GeneRuler Ultra Low Range DNA Ladder, #SM1211/2/3, p.417
OGeneRuler Ultra Low Range DNA Ladder, #SM1223, p.417
GeneRuler Low Range DNA Ladder, #SM1191/2/3, p.417
OGeneRuler Low Range DNA Ladder, #SM1203, p.417
GeneRuler 50bp DNA Ladder, #SM0371/2/3, p.416
OGeneRuler 50bp DNA Ladder, #SM1133, p.416
GeneRuler 1kb Plus DNA Ladder, #SM1331/2/3/4, p.416
OGeneRuler 1kb Plus DNA Ladder, #SM1343, p.416
GeneRuler 100bp DNA Ladder, #SM0241/2/3/4, p.416
OGeneRuler 100bp DNA Ladder, #SM1143, p.416
GeneRuler 100bp Plus DNA Ladder, #SM0321/2/3/4, p.416
OGeneRuler 100bp Plus DNA Ladder, #SM1153, p.416
GeneRuler Express DNA Ladder, #SM1551/2/3, p.417
OGeneRuler Express DNA Ladder, #SM1563, p.417
GeneRuler DNA Ladder Mix, #SM0331/2/3/4, p.416
OGeneRuler DNA Ladder Mix, #SM1173, p.416
GeneRuler 1kb DNA Ladder, #SM0311/2/3/4, p.416
OGeneRuler 1kb DNA Ladder, #SM1163, p.416
GeneRuler High Range DNA Ladder, #SM1351/2/3, p.417
80-1031bp
100-1000bp
100-1000bp
MassRuler Low Range DNA Ladder, #SM0383, p.419

MassRuler Express LR Forward DNA Ladder, #SM1263, p.419
MassRuler Express LR Reverse DNA Ladder, #SM1273, p.419
80-10000bp
100-10000bp
100-10000bp
MassRuler DNA Ladder Mix, #SM0403, p.419

MassRuler Express Forward DNA Ladder Mix, #SM1283, p.419
MassRuler Express Reverse DNA Ladder Mix, #SM1293, p.419
MassRuler High Range DNA Ladder, #SM0393, p.419
MassRuler Express HR Forward DNA Ladder, #SM1243, p.419
MassRuler Express HR Reverse DNA Ladder, #SM1253, p.419
1500-10000bp
1500-10000bp
1500-10000bp
500-10000bp
FastRuler Ultra Low Range DNA Ladder, #SM1233, p.420

FastRuler Low Range DNA Ladder, #SM1103, p.420
FastRuler Middle Range DNA Ladder, #SM1113, p.420
FastRuler High Range DNA Ladder, #SM1123, p.420
10-100bp
10-150bp
20-300bp
50-1000bp
100-1500bp
100-6000bp
200-3000bp
500-6000bp
ORangeRuler 5bp DNA Ladder, #SM1303, p.423

ORangeRuler 100+500bp DNA Ladder, #SM0653, p.423
10-200bp
50-1500bp
100-5000bp
ZipRuler Express DNA Ladder 1, #SM1373, p.421

ZipRuler Express DNA Ladder 2, #SM1373, p.421
100-10000bp
200-20000bp
pBR322 DNA/BsuRI Marker, 5, #SM0271, p.426

pBR322 DNA/AluI Marker, 20, #SM0121/3, p.427
Lambda DNA/PstI Marker, 24, #SM0361, p.425
pUC Mix Marker, 8, #SM0301/2/3, p.427
Lambda DNA/Eco47I Marker, 13, #SM1051, p.425
X174 DNA/HinfI Marker, 10, #SM0261, p.427
pUC19 DNA/MspI Marker, 23, #SM0221/2/3, p.427
X174 DNA/BsuRI Marker, 9, #SM0251/2/3, p.427
Lambda pUC Mix Marker, 4, #SM0291, p.425
Lambda DNA/HindIII Marker, 2, #SM0101/2/3, p.425
Lambda DNA/EcoRI+HindIII Marker, 3, #SM0191/2/3, p.425
Lambda Mix Marker, 19, #SM0231, p.425
Lambda DNA/EcoRI Marker, 1, #SM0281, p.425
8-587bp
11-908bp
15-11501bp
19-1118bp
23-8126bp
24-726bp
26-501bp
72-1353bp
74-19329bp
74-19329bp
117-8453bp
125-23130bp
125-21226bp
1503-48502bp
3530-21226bp
10bp
100bp
1kb
10kb
50kb
Bulk quantities and custom formulations available upon request
409
410
50kb
www.fermentas.com
FastRuler High Range
DNA Ladder
#SM1123, p.420
FastRuler Middle Range

DNA Ladder
#SM1113, p.420
FastRuler Low Range

DNA Ladder
#SM1103, p.420
FastRuler Ultra Low Range

DNA Ladder
#SM1233, p.420
MassRuler Express HR Forward

DNA Ladder #SM1243, p.419
MassRuler Express HR Reverse
MassRuler High Range

DNA Ladder
#SM0393, p.419
MassRuler Express Forward

DNA Ladder Mix #SM1283, p.419
MassRuler Express Reverse
DNA Ladder Mix #SM1293, p.419
MassRuler DNA
Ladder Mix
#SM0403, p.419
MassRuler Express LR Forward

MassRuler Express LR Reverse
GeneRuler High Range

DNA Ladder
#SM1351/2/3, p.417
GeneRuler 1kb DNA Ladder

#SM0311/2/3,p.416OGeneRuler
1kb DNA Ladder
#SM1163, p.416
GeneRuler DNA Ladder Mix

#SM0331/2/3, p.416
OGeneRuler DNA Ladder Mix
#SM1173, p.416
GeneRuler Express DNA Ladder

#SM1551/2/3, p.417
OGeneRuler Express DNA Ladder
#SM1563 p.417
GeneRuler 100bp Plus DNA Ladder

#SM0321/2/3, p.416
OGeneRuler 100bp Plus DNA Ladder
#SM1153 p.416
GeneRuler 100bp DNA Ladder

#SM0241/2/3 p.416
OGeneRuler 100bp DNA Ladder
#SM1143 p.416
GeneRuler 1kb Plus DNA Ladder

#SM1331/2/3, p.416
OGeneRuler 1kb Plus DNA Ladder
#SM1343, p.416

#SM0371/2/3, p.416
OGeneRuler 50bp DNA Ladder
#SM133, p.416
GeneRuler Low Range DNA Ladder

#SM1191/2/3, p.417
OGeneRuler Low Range DNA Ladder
#SM1203, p.417
GeneRuler Ultra Low Range

DNA Ladder #SM1211/2/3, p.417
OGeneRuler Ultra Low Range
50kb
MassRuler Low Range

DNA Ladder
#SM0383, p.419
Reference for DNA Ladders and Markers
50kb
10kb
10kb
1kb
1kb
100bp
100bp
10bp
10bp
50kb
10kb
10kb
1kb
1kb
100bp
100bp
10bp
10bp
50kb
10bp
X174 DNA/BsuRI, 9
#SM0251/2/3, p.427
pUC19 DNA/MspI, 23
#SM0221/2/3, p.427
X174 DNA/HinfI, 10
#SM0261, p.427
Lambda DNA/Eco47I, 13
#SM1051, p.425
pUC Mix, 8
#SM0301/2/3, p.427
Lambda DNA/PstI, 24
#SM0361, p.425
pBR322 DNA/AluI, 20
#SM0121/3, p.427
pBR322 DNA/BsuRI, 5
#SM0271, p.426
50kb
ZipRuler Express DNA
Ladder 2
#SM1373, p.421
ZipRuler Express DNA

Ladder 1
#SM1373, p.421
ORangeRuler 500bp
DNA Ladder
#SM0643, p.423
ORangeRuler 200bp
DNA Ladder
#SM0633, p.423
ORangeRuler 100+500bp
DNA Ladder
#SM0653, p.423
ORangeRuler 100bp
DNA Ladder
#SM0623, p.423
ORangeRuler 50bp
DNA Ladder
#SM0613, p.423
ORangeRuler 20bp
DNA Ladder
#SM1323, p.423
ORangeRuler 10bp
DNA Ladder
#SM1313, p.423
ORangeRuler 5bp
DNA Ladder
#SM1303, p.423
100bp
* Indicates the fragments with cohesive ends of the 12 nt cos sites of DNA that may anneal and form an additional band.
These fragments (shown in italic) can be separated by heating at 65C for 5min and then cooling on ice for 3min.
Indicates a possible additional band due to annealing of cohesive ends of the fragments from 12 nt cos sites of DNA.
Reference bands appear in red.
50kb
10kb
10kb
1kb
1kb
100bp
100bp
10bp
10bp
50kb
10kb
10kb
1kb
1kb
100bp
10bp
411
10kb
1kb
100bp
10bp
Lambda DNA/EcoRI, 1
#SM0281, p.425
Lambda Mix, 19
#SM0231, p.425
Lambda DNA/EcoRI+HindIII, 3
#SM0191/2/3, p.425
Lambda DNA/HindIII, 2
#SM0101/2/3, p.425
#SM0111, p.425
#SM0161, p.425
Lambda pUC Mix, 4

#SM0291, p.425
50kb
50kb
10kb
1kb
100bp
10bp
* Indicates the fragments with cohesive ends of the 12 nt cos sites of DNA that may anneal and form an additional band.
These fragments (shown in italic) can be separated by heating at 65C for 5min and then cooling on ice for 3min.
Indicates a possible additional band due to annealing of cohesive ends of the fragments from 12 nt cos sites of DNA.
Reference bands appear in red.
www.fermentas.com
412
Products
NoLimits Individual DNA Fragments and Custom DNA Ladders
Collection of Individual DNA Fragments
DNA fragment
size
10bp
15bp
20bp
25bp
35bp
50bp
75bp
100bp
150bp
200bp
250bp
300bp
400bp
500bp
600bp
700bp
SM1391
SM1381
SM1401
SM1761
SM1411
SM1421
SM1431
SM1441
SM1601
SM1611
SM1451
SM1621
SM1631
SM1641
SM1461
SM1651
Amount,
g
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
750bp
SM1471
10
Features
800bp
SM1481
10
850bp
SM1661
10
900bp
SM1491
10
1000bp
SM1671
10
1200bp
SM1681
10
1500bp
SM1691
10
Chromatography-purified individual DNA

fragments.
Broad range 10-20000bp.
Sharp peaks during capillary electrophoresis,
sharp bands after gel electrophoresis.
Precise DNA concentration.
2000bp
SM1701
10
2500bp
SM1571
10
3000bp
SM1711
10
3500bp
SM1501
10
4000bp
SM1721
10
5000bp
SM1731
10
6000bp
SM1511
10
7000bp
SM1741
10
8000bp
SM1521
10
10000bp
SM1751
10
15000bp
SM1531
10
17000bp
SM1771
10
20000bp
SM1541
10
Cat. #
Description
NoLimits is a collection of 36 highlypurified
individual DNA fragments ranging from
10 to 20,000bp.
NoLimits DNA fragments are produced using
specifically designed plasmids purified by a
patented technology. Plasmid DNA is digested
with PureExtreme restriction enzymes and the
individual DNA fragments are chromatographypurified from the digestion mixture. As a result,
we offer exceptionally pure DNA fragments
that are free of any truncated or degraded
molecules. Our custom DNA fragments are
the best choice for applications ranging from
electrophoresis (both gel and capillary) to HPLC
and beyond.
Applications
Gel electrophoresis.
Capillary electrophoresis.
DNA quantification.
HPLC analysis.
Applications where a custom DNA size
standard is required.
Concentration
0.5g/l
Storage Buffer (TE buffer)

10mM Tris-HCl (pH7.6) with 1mM EDTA.
Quality Control
Tested on an Agilent 2100 bioanalyzer and by
appropriate gel electrophoresis. DNA concentration
determined spectrophotometrically. The absence of
nucleases confirmed by appropriate tests.
Storage
Store at -20C.
1.1%TopVision Agarose (#R0491), 1X TAE, 7 V/cm, 30min.

NoLimits DNA fragments from 300bp to 10kb.
NoLimits Custom DNA Ladders Service

Bulk orders can be customized to fit your needs:
Individual DNA fragments or mixtures.
Custom ladders of any combination and
concentration.
Reference bands in a custom-made DNA ladder.
Flexible formulations.
Readytouse versions premixed with any
loading dye (see p.428).
From microgram to gram quantities.
How to Order?
Contact your local Fermentas representative:
see Contacts on www.fermentas.com
413
GeneRuler and OGeneRuler DNA Ladders (10-20000bp)

Related Products
TopVision Agaroses
Loading Dyes
50X TAE Buffer
10X TBE Buffer
T4 Polynucleotide Kinase
ATP
0.5 M EDTA, pH8.0
Water, nuclease-free
p.484
p.428
p.430
p.430
p.244
p.469
p.477
p.476
Description
Features
GeneRuler DNA ladders are mixtures of chromatography-purified individual DNA fragments.

The GeneRuler line includes the most popular
ladders such as the 100bp and 1kb DNA
Ladder.
GeneRuler DNA ladders are available in
two formats: conventional (TE buffer) and a
readytouse format (premixed with 6X DNA
Loading Dye which contains bromophenol blue
and xylene cyanol FF).
Conventional versions can be labeled radioactively with T4 Polynucleotide Kinase (#EK0031).
OGeneRuler DNA ladders are another
readytouse version of GeneRuler DNA ladders. They are premixed with 6X Orange DNA
Loading Dye, which contains xylene cyanol FF
and orange G. In a 1%agarose gel orange G
dye migrates at 50bp. Therefore OGeneRuler
DNA ladders are ideal when visualization of
small DNA fragments is important.
GeneRuler Ultra Low Range and Low
Range DNA ladders are ideal for denaturing
polyacrylamide gel electrophoresis (Fig.9.4).
GeneRuler and OGeneRuler Ultra Low
Range DNA ladders are ideal for analysis of
siRNA.
GeneRuler High Range DNA Ladder is
designed for fast sizing of high molecular weight
DNA fragments (1.5 h in 0.4%agarose gel).
GeneRuler and OGeneRuler Express
DNA ladders are designed for fast separation
(in 5-15min at 23 V/cm) under a wide range of
electrophoresis conditions, in different buffers,
voltages or gel percentages.
The ladders are supplied (depending on the version) either with 6X DNA Loading Dye or with 6X
Orange DNA Loading Dye for sample DNA.
Ideal for both DNA sizing and approximate

quantification.
Sharp bands.
Assembled of chromatography purified
individual DNA fragments.
Bright reference bands.
Readytouse ladders can be directly loaded
and are stable at room temperature for
6months.
Supplied with loading dye for sample DNA.

10mM Tris-HCl (pH7.6) and 1mM EDTA.
GeneRuler Storage and Loading Buffer

(for readytouse ladders)
10mM Tris-HCl (pH7.6), 10mM EDTA, .
0.005%bromophenol blue, 0.005%xylene
cyanol FF and 10%glycerol.
6X DNA Loading Dye

10mM Tris-HCl (pH7.6), 0.03%bromophenol
blue, 0.03%xylene cyanol FF, 60%glycerol and
60mM EDTA.
OGeneRuler Storage and Loading Buffer

0.025%orange G, 0.005%xylene cyanol FF
and 10%glycerol.
6X Orange DNA Loading Dye

10mM Tris-HCl (pH7.6), 0.15%orange G,
0.03%xylene cyanol FF, 60%glycerol and .
60mM EDTA.
Quality Control
Tested in appropriate gel electrophoresis applications. Concentration of each DNA fragment
and of the complete ladder determined spectrophotometrically. The absence of nucleases
confirmed by appropriate tests.
Storage
Store at -20C.
Readytouse versions can be stored at room
temperature or at 4C for 6months.
Protocols and Recommendations

9.1. General recommendations for DNA
electrophoresis
9.2. Preparation of agarose gels for DNA
electrophoresis
9.3. Preparation of gels for PAGE
9.4. Preparation of DNA samples for
electrophoresis
9.5. Preparation of DNA ladders/markers
for electrophoresis
9.7. Separation of Express DNA ladders in
different electrophoresis conditions
7.1. DNA/RNA end labeling
www.fermentas.com
414
p.431
p.431
p.432
p.433
p.433
p.434
p.380
DNA ladder
Cat. #

GeneRuler 1kb DNA Ladder,
readytouse
OGeneRuler 1kb DNA Ladder,
readytouse
SM0311.
SM0312
SM0313
SM0314
0.5
0.1
SM1163
SM1331.
SM1332
SM1333
SM1334
GeneRuler 1kb Plus DNA Ladder,

readytouse
OGeneRuler 1kb Plus DNA Ladder,
SM1343
readytouse
SM0331.
SM0332
GeneRuler DNA Ladder Mix,
SM0333
readytouse
SM0334
OGeneRuler DNA Ladder Mix,
SM1173
readytouse
SM0241.
SM0242
GeneRuler 100bp DNA Ladder,
SM0243
readytouse
SM0244
OGeneRuler 100bp DNA Ladder,
SM1143
readytouse
SM0321.
SM0322
GeneRuler100bp Plus DNA Ladder, SM0323
readytouse
SM0324
OGeneRuler 100bp Plus DNA
SM1153
Ladder, readytouse
SM0371.
SM0372
GeneRuler 50bp DNA Ladder,
SM0373
readytouse

SM1133
readytouse
GeneRuler Ultra Low Range DNA

SM1211.
Ladder
SM1212
GeneRuler Ultra Low Range DNA
SM1213
Ladder, readytouse
OGeneRuler Ultra Low Range DNA

SM1223
Ladder, readytouse
SM1191.
SM1192
GeneRuler Low Range DNA Ladder,
SM1193
readytouse
OGeneRuler Low Range DNA

SM1203
Ladder, readytouse
SM1351.
GeneRuler High Range DNA Ladder
SM1352
GeneRuler High Range DNA Ladder,
SM1353
readytouse
SM1551.
SM1552
GeneRuler Express DNA Ladder,
SM1553
readytouse
OGeneRuler Express DNA Ladder,

SM1563
readytouse
Supplied with:
6X DNA Loading Dye
Concentration,
g/l
0.5
0.1
Amount,
g
Applications, Loading,
0.5g/lane g(l)/lane
250 (5x50).
1250 (25x50)
250 (5x50)
50
500.
2500
500
100
250 (5x50)
500
250 (5x50).
1250 (25x50)
250 (5x50)
50
500.
2500
500
100
250 (5x50)
500
50.
250 (5x50)
50
250 (5x50
100.
500
100
500
50
100
50.
250 (5x50)
50
250 (5x50)
100.
500
100
500
50
100
0.5
50.
250 (5x50)
100.
500
0.5 (1)
0.1
50
100
0.5 (5)
0.5
50.
250 (5x50)
50-100.
250-500
0.5-1 (1-2)
0.5
0.1
14
0.7-1.2
75-20000
15
0.7-1.2
100-10000
21
0.7-1.2
100-1000
10
1.7-2.5
4-8
100-3000
14
1.7-2.5
50-1000
13
1.7-2.5
4-8
10-300
11
4.5-5.0
8-10
25-700
10
2.5-3.0
8-10
1017148502
0.3-0.5
100-5000
1.7-2.5
0.5 (5)
500
0.1
250-10000
0.5 (1)
500.
2500
500
100
0.5
PAGE,
%
0.5 (5)
250 (5x50).
1250 (25x50)
250 (5x50)
50
0.1
Number of Agarose,
fragments
%
0.5 (1)
250 (5x50)
0.5
Range,
bp
0.5 (1)
0.5 (5)
0.5 (1)
0.5 (5)
0.5 (1)
0.5 (5)
0.1
50
100
0.5-1 (5-10)
0.5
50.
250 (5x50)
50-100.
250-500
0.5-1 (1-2)
0.1
50
100
0.5-1 (5-10)
0.5
50.
250 (5x50)
100.
500
0.5 (1)
0.1
50
100
0.5 (5)
0.5
50.
250 (5x50)
100.
500
0.5 (1)
0.1
50
100
0.5 (5)
1ml/2ml/10ml
1ml/2ml/10ml
415
#SM1163
10000
8000
6000
5000
4000
3500
3000
2500
2000
1500
#SM1343
#SM1173
bp ng/0.5g
30.0 6.0
30.0 6.0
70.0 14.0
30.0 6.0
30.0 6.0
30.0 6.0
70.0 14.0
25.0 5.0
25.0 5.0
25.0 5.0
20000
10000
7000
5000
4000
3000
1000 60.0 12.0

750 25.0 5.0
500
25.0
5.0
250
25.0
5.0
bp ng/0.5g
20.0 4.0
20.0 4.0
20.0 4.0
75.0 15.0
20.0 4.0
20.0 4.0
2000 20.0 4.0

1500 80.0 16.0
1000
700
500
400
300
200
75
25.0 5.0
25.0 5.0
75.0 15.0
25.0 5.0
25.0 5.0
25.0 5.0
25.0 5.0
0.5g/lane, 8cm length gel,

1X TAE, 7 V/cm, 45min
#SM0241/2/3
#SM0321/2/3

readytouse
OGeneRuler 100bp Plus DNA Ladder,

readytouse
45.0 9.0
45.0 9.0
45.0 9.0
45.0 9.0
45.0 9.0
115.0 23.0
40.0 8.0
40.0 8.0
200
40.0
8.0
100
40.0
8.0

1X TBE, 5 V/cm, 1 h
1000
900
800
700
600
500
400
300
200

1X TAE, 8 V/cm, 3 h
www.fermentas.com
3000
2000
1500
1200
1000
900
800
700
600
500
400
28.0 5.6
28.0 5.6
28.0 5.6
28.0 5.6
80.0 16.0
27.0 5.4
27.0 5.4
27.0 5.4
27.0 5.4
80.0 16.0
30.0 6.0
300
30.0
6.0
200
30.0
6.0
100
30.0
6.0

1X TBE, 5 V/cm, 1 h
100

readytouse
#SM1133
bp ng/0.5g
1.7%TopVision Agarose (#R0491)
1000
900
800
700
600
500
400
300
#SM1153
bp
5%polyacrylamide
18.0 3.6
18.0 3.6
18.0 3.6
18.0 3.6
18.0 3.6
18.0 3.6
60.0 12.0
16.0 3.2
16.0 3.2
16.0 3.2
16.0 3.2
60.0 12.0
17.0 3.4
17.0 3.4
17.0 3.4
17.0 3.4
60.0 12.0
20.0 4.0
20.0 4.0
20.0 4.0
20.0 4.0
#SM0371/2/3
bp
bp ng/0.5g
3000
2000
1500
1200
1000
900
800
700
600
500
400
300
200
5%polyacrylamide
#SM1143
10000
8000
6000
5000
4000
3500
3000
2500
2000
1500
1200
1000
900
800
700
600
500
400
300
200
100


bp ng/0.5g
416
OGeneRuler DNA Ladder Mix,

readytouse
1%TopVision Agarose (#R0491)
bp ng/0.5g
#SM0331/2/3
OGeneRuler 1kb Plus DNA Ladder,

readytouse
1000
900
800
700
600
500
400
300
250
200
150
100
30.0 6.0
30.0 6.0
30.0 6.0
30.0 6.0
30.0 6.0
75.0 15.0
30.0 6.0
30.0 6.0
75.0 15.0
35.0 7.0
35.0 7.0
35.0 7.0
50
35.0

1X TBE, 5 V/cm, 1 h
100

1X TAE, 8 V/cm, 3 h
bp
1000
900
800
700
600
500
400
300
250
200
7.0
150
100
5%polyacrylamide
OGeneRuler 1kb DNA Ladder,

readytouse
#SM1331/2/3
#SM0311/2/3
50

1X TAE, 8 V/cm, 3 h
GeneRuler Ultra Low Range DNA Ladder
#SM1211/2/3
#SM1191/2/3
OGeneRuler Ultra Low Range DNA Ladder,

readytouse
OGeneRuler Low Range DNA Ladder,

readytouse
#SM1223
#SM1203
30.0
32.5
35.0
6.0
6.5
7.0
300
100
75
37.5
37.5
7.5
7.5
200
150
50
35
25
20
15
10
bp ng/0.5g
700
500
400
300
30.0 6.0
32.5 6.5
35.0 7.0
90.0 18.0
35.0
35.0
105.0 21.0
37.5 7.5
37.5 7.5
40.0 8.0
47.5 9.5
100
75
200
150
50
100
75
90.0 18.0
37.5 7.5
60.0 12.0
35
50
40.0
25
75.0 15.0
25
20
15
10%polyacrylamide

1X TBE, 5 V/cm, 1 h
10

1X TBE, 5 V/cm, 1 h
bp ng/0.5g
bp
700
500
400
300
200
150
7.0
7.0
100
75
50
8.0
25
300
200
150
#SM1351/2/3
48502
24508
20555
17000
15258
13825
12119
80.0
88.0
80.0
59.0
59.0
50.0
47.0
16.0
17.7
15.9
11.8
11.8
10.0
9.4
10171 37.0
7.3

1X TAE, 3 V/cm, 1.5 h
10%polyacrylamide
bp ng/0.5g
bp
GeneRuler High Range DNA Ladder
0.5g/lane, 2 0cm length gel,

1X TBE, 8 V/cm, 3 h
0.5g/lane, 20c m length gel,

1X TBE, 8 V/cm, 3 h

#SM1551/2/3
OGeneRuler Express DNA Ladder,

readytouse
#SM1563
bp ng/0.5g
5000 40.0
8.0
3000 40.0
8.0
5min
10min
15min
2000 40.0 8.0

1500 100.0 20.0
1000 40.0
750 40.0
8.0
8.0
500 100.0 20.0

300
50.0 10.0
100
50.0 10.0

Figure 9.1. Fast separation of GeneRuler

Express DNA Ladder at high voltage (23 V/cm).
Electrophoresis conditions: 0.5g/lane, 1%TopVision
Agarose (#R0491), 1X TAE, 23 V/cm, 5-15min.
417
MassRuler DNA Ladders, readytouse (80-10000bp)

DNA ladder, readytouse
MassRuler Low Range DNA Ladder
MassRuler Express LR Forward DNA Ladder
MassRuler Express LR Reverse DNA Ladder
MassRuler High Range DNA Ladder
MassRuler Express HR Forward DNA Ladder
MassRuler Express HR Reverse DNA Ladder
MassRuler DNA Ladder Mix
MassRuler Express Forward DNA Ladder Mix
MassRuler Express Reverse DNA Ladder Mix
Supplied with:
6X MassRuler DNA Loading Dye
SM0383
SM1263
SM1273
SM0393
SM1243
SM1253
SM0403
SM1283
SM1293
Volume,
l
Applications
Loading,
l/lane
2x500
50-200
5-20
2x500
50-200
5-20
2x500
50-200
5-20
60.8
28
Range,
bp
Number of Agarose,
fragments
%
80-1031
11
1.7-2.5
100-1000
0.7-2.0
0.7-1.2
0.7-1.5
80-10000
20
1.0-1.2
100-10000
12
0.7-1.5
42.2
28.5
103
56.5
1500-10000
1ml
Related Products

Concentration,
ng/l
Cat. #
TopVision Agaroses
Loading Dyes
50X TAE Buffer
10X TBE Buffer
p.484
p.428
p.430
p.430
p.476
Description
Features
MassRuler DNA ladders are specially designed

for accurate quantification and sizing of DNA
fragments by agarose gel electrophoresis. The
intensity of the fragment in each ladder is calibrated against a standard that guarantees the
precise quantity of each band. The ladders are
mixtures of chromatography-purified, individual
DNA fragments.
MassRuler Express Forward and Reverse DNA
Ladders allow fast and reliable DNA quantification in short separation distances under various
electrophoresis conditions. In the forward
versions, the mass of each DNA fragment
is directly proportional to the fragment size,
whereas in the reverse versions, the mass is
inversely proportional to the fragments size.
Accurate DNA sizing and quantification.

Sharp bands.
Easy-to-remember fragment sizes and
quantities.
Readytouse premixed with Loading Dye.
Storage and Loading Buffer

0.005%bromophenol blue, 10%glycerol.

blue, 60%glycerol and 60mM EDTA.
Quality Control
Storage
Store at -20C (or at 4C or room temperature
for 6 months).
A. MassRuler Express LR Forward DNA Ladder

[FU]
5000

electrophortesis
p.431
9.1.1. Recommendations for accurate in-gel
quantification
p.431
electrophoresis
p.431
electrophoresis
p.433
9.7. Separation of Express DNA ladders
in different electrophoresis conditions
p.434
www.fermentas.com
418
B. MassRuler Express LR Reverse DNA Ladder

[FU]
5000
4000
4000
3000
3000
2000
2000
1000
1000
0
40
60
80
100
40
60
80
100
Figure 9.2. Analysis of the MassRuler Express LR Forward and Reverse DNA Ladders (#SM1263 and
#SM1273) using the Agilent 2100 Bioanalyzer DNA 1000 LabChip Kit.
A analysis of MassRuler Express LR Forward DNA Ladder
B analysis of MassRuler Express LR Reverse DNA Ladder
MassRuler Low Range DNA Ladder,
readytouse
MassRuler Express LR Forward DNA Ladder, readytouse
#SM0383
MassRuler Express LR Reverse DNA Ladder, readytouse
#SM1263
#SM1273
bp ng/20 l ng/15 l ng/10 l ng/5 l
ng/20 l ng/15 l ng/10 l ng/5 l bp
Forward Reverse
1031
900
800
700
600
500
400
200
180
160
140
120
200
80
150
135
120
105
90
150
60
100
90
80
70
60
100
40
50
45
40
35
30
50
20
300
60
45
30
15
200
40
30
20
100
80
20
16
15
2
10
8
200
150
100
50
1000
1000
20
15
10
10
140
100
105
75
70
50
35
25
700
500
700
500
40
60
30
45
20
30
10
15
5
4
60
40
20
45
30
15
30
20
10
15
10
5
300
200
100
300
200
100
100
140
200
75
105
150
50
70
100
25
35
50
10 l/lane, 8cm length gel,

1X TBE, 5 V/cm, 1.5 h

10 l/lane, 8cm length gel, 1X TAE, 7 V/cm, 30min
MassRuler High Range DNA Ladder,

readytouse
MassRuler Express HR Forward DNA Ladder, readytouse
#SM0393
MassRuler Express HR Reverse DNA Ladder, readytouse
#SM1243
#SM1253
Forward Reverse
10000
8000
6000
5000
4000
3000
2500
2000
1500
200
160
120
100
80
60
52
40
32
150
120
90
75
60
45
39
30
24
100
80
60
50
40
30
26
20
16
50
40
30
25
20
15
13
10
8
200
140
100
150
105
75
100
70
50
50 10000
35 7000
25 5000
10000
7000
5000
30
40
60
22.5
30
45
15
20
30
7.5
10
15
60
45
30
15
3000
3000
100
75
50
25
40
30
30
22.5
20
15
10
7.5
2000
1500
2000
1500
140
200
105
150
70
100
35
50


MassRuler DNA Ladder Mix,

readytouse
MassRuler Express Forward DNA Ladder Mix, readytouse
#SM0403
MassRuler Express Reverse DNA Ladder Mix, readytouse
#SM1283
#SM1293
Forward Reverse
10000
8000
6000
5000
4000
3000
2500
2000
1500
1031
900
800
700
600
500
400
300
200
100
80
200
160
120
100
80
60
52
40
32
200
180
160
140
120
200
80
60
40
20
16

150
120
90
75
60
45
39
30
24
150
135
120
105
90
150
60
45
30
100
80
60
50
40
30
26
20
16
100
90
80
70
60
100
40
30
20
50
40
30
25
20
15
13
10
8
50
45
40
35
30
50
20
15
10
15
10
12 8
5
4
200
140
100
150
105
75
100
70
50
50 10000
35 7000
25 5000
10000
7000
5000
30
40
60
22.5
30
45
15
20
30
7.5
10
15
60
40
30
45
30
22.5
30
20
15
15
10
7.5
3000
2000
1500
3000
2000
1500
100
140
200
75
105
150
50
70
100
25
35
50
200
150
100
50
1000
1000
20
15
10
140
100
105
75
70
50
35
25
700
500
700
500
40
60
30
45
20
30
10
15
60
40
20
45
30
15
30
20
10
15
10
5
300
200
100
300
200
100
100
140
200
75
105
150
50
70
100
25
35
50

419
FastRuler DNA Ladders, readytouse (10-10000bp)

FastRuler Ultra Low Range DNA Ladder
FastRuler Low Range DNA Ladder
FastRuler Middle Range DNA Ladder
FastRuler High Range DNA Ladder
Supplied with:
Concentration,
ng/l
SM1233
SM1103
SM1113
SM1123
22.2
20
20
20
Volume,
l
Applications
2x500
50-333
Loading,
l/lane
Range,
bp
TopVision Agaroses
Loading Dyes
50X TAE Buffer
10X TBE Buffer
ATP
0.5 M EDTA, pH8.0
p.484
p.428
p.430
p.430
p.244
p.469
p.477
p.476
3-20
10-200
50-1500
100-5000
500-10000
FastRuler DNA ladders are specifically

designed for fast sizing and quantification of
double-stranded DNA in 48 well (or96-well)
high throughput gels, as well as in conventional
agarose gels. These ladders are mixtures of five
blunt-end chromatography-purified individual
DNA fragments that are easily resolved in a
short separation distance (10-20mm) after an
8-14min run. The ladders are especially useful
for electrophoresis of PCR products. FastRuler
Ultra Low Range DNA Ladder, readytouse contains dephosphorylated DNA fragments and is
ideal for 5-end labeling with T4 Polynucleotide
Kinase (#EK0031) in a forward reaction.


(for FastRuler Ultra Low Range)
0.025%orange G, 0.005%xylene cyanol FF,
10%glycerol.

blue, 60%glycerol and 60mM EDTA.

60mM EDTA.
Fast separation (8-14min).

Short separation distance (10-20mM).
Sharp bands.
Easy-to-remember fragment sizes and
quantities.
Readytouse premixed with loading dye.
Supplied with loading dye solution for sample
DNA.
Quality Control
FastRuler Ultra Low Range DNA Ladder,

readytouse
FastRuler Low Range DNA Ladder,

readytouse
#SM1233

Storage
for 6 months).
#SM1103
bp ng/20 l ng/15 l ng/10 l ng/5 l ng/3 l
200
100
50
20
10
80
80
80
80
124
60
60
60
60
93
40
40
40
40
62
20
20
20
20
31
bp ng/20 l ng/15 l ng/10 l ng/5 l n g/3 l

1500
80
60
40
20
12
850
80
60
40
20
12
400
80
60
40
20
12
200
80
60
40
20
12
50
80
60
40
20
12
12
12
12
12
19

20 l/lane, 1X TBE, 7 V/cm, 14min

electrophoresis
electrophoresis
electrophoresis
for electrophoresis
7.1.2. DNA/RNA 5-end labeling by
T4 PNK in the forward reaction
420
4.0
2.0
1.0
1.0
Description
Features
www.fermentas.com
Number of Agarose,
fragments
%
1ml
1ml
Related Products

Cat. #
p.431
p.431
p.433
p.433
p. 380

20 l/lane, 1X TBE, 7 V/cm, 14min
FastRuler High Range DNA Ladder,

readytouse
FastRuler Middle Range DNA Ladder,

readytouse
#SM1123
#SM1113
bp ng/20 l ng/15l ng/10 l ng/5 l ng/3 l
bp ng/20 l ng/15 l ng/10 l ng/5 l ng/3 l
5000
2000
10000
4000
2000
1000
500
80
80
60
60
40
40
20
20
12
12
850
80
60
40
400
80
60
40
100
80
60
40
20 l/lane, 1X TAE, 7 V/cm, 14min
20
20
20
12
12
12
80
80
80
80
80
60
60
60
60
60
40
40
40
40
40
20
20
20
20
20
12
12
12
12
12

20 l/lane, 1X TAE, 7 V/cm, 14min
ZipRuler Express DNA Ladder Set, readytouse (100-20000bp)

Cat. #
ZipRuler Express DNA Ladder Set

ZipRuler Express DNA Ladder 1
ZipRuler Express DNA Ladder 2
Supplied with:
SM1373
0.1
2X50
100-200
Loading,
g(l)/lane
Range,
bp
Number of Agarose,
fragments
%
0.25-0.5 (2.5-5)
100-10000.
200-20000
5.0
1ml
Description
Related Products

Concentration, Amount,
Applications
g/l
g
TopVision Agaroses
Loading Dyes
50X TAE Buffer
10X TBE Buffer

electrophoresis
9.2. Preparation of agarose gels for
DNA electrophoresis
electrophoresis
9.7. Separation of Express DNA ladders
in different electrophoresis conditions

(for ZipRuler Express DNA Ladder 1)
The ZipRuler Express DNA Ladder Set contains

two ladders: ZipRuler Express DNA Ladder 1
and ZipRuler Express DNA Ladder 2. Both are
mixtures of chromatography-purified individual
DNA fragments. The set is designed for fast and
accurate sizing of a broad range of DNA fragments and allows electrophoresis at different
conditions.
For fast separation, the two ladders are loaded
into two different wells of the same gel. For
longer runs, equal amounts of both ladders are
loaded into the same gel well.
ZipRuler Express DNA Ladder 1 is premixed
with the 6X Orange DNA Loading Dye (orange
G and xylene cyanol), whereas ZipRuler
Express DNA Ladder 2 is premixed with the 6X
MassRuler DNA Loading Dye (bromophenol
blue). When both ladders are loaded in the same
gel lane, the migration of DNA is monitored by
three electrophoresis tracking dyes (bromophenol blue, xylene cyanol FF and orange G).
p.484
p.428
p.430
p.430
p.476

10%glycerol.

(for ZipRuler Express DNA Ladder 2)

60mM EDTA.
Quality Control
Storage
Store -20C (or at 4C or at room temperature
for up to 6 months).
Features
Flexible - can be run in two lanes or combined in one lane.
Readytouse premixed with a loading dye for
direct loading and room temperature storage.
Supplied with a loading dye for sample DNA.
p.431
p.431
p.433
p.434
ZipRuler Express DNA Ladder Set, readytouse,

#SM1373
% ng/0.5g bp
8.8 44 10000
25.6 128 5000
8.8 44 3000
8.8 44 2000
8.0 40 1200
8.0 40 850
16.0 80 500
8.0 40 300
8.0 40 100
20min
bp ng/0.5g %
20000 48 9.6
7000 48 9.6
4000 46 9.2
2500 46 9.2
1500 152 30.4
1000 40 8.0
700
40 8.0
400
40 8.0
200
40 8.0
Mix
% ng/0.5g bp
8.8 44 10000
25.6 128 5000
20min

8.8
8.8
44 3000
44 2000
8.0
8.0
40 1200
40 850
16.0
80
8.0
40
300
8.0
40
100
bp ng/0.5g %
20000 48
7000 48
9.6
9.6
4000
46
9.2
2500
46
9.2
Mix
20000
10000
7000
5000
4000
3000
2500
2000
1500
1200
1000
850
700
500
400
300
200
100
1500 152 30.4
500
40min
1000
700
40
40
8.0
8.0
400
40
8.0
200
40
8.0
bp
40min

1 ZipRuler Express
DNA Ladder 1
2 ZipRuler Express
DNA Ladder 2
Mix Ladder 1 and
Ladder 2 mixed in
equal volumes
421
ORangeRuler DNA Ladders, readytouse (10-6000bp)

Cat. #
ORangeRuler 5bp DNA Ladder

ORangeRuler 100+500bp DNA
Ladder
Supplied with:
SM1303
SM1313
SM1323
SM0613
SM0623
SM0633
SM0643
0.1
0.05
50
25
50-100
100
0.5-1 (5-10)
0.25 (5)
SM0653
10-100
10-150
20-300
50-1000
100-1500
200-3000
500-6000
19
15
15
20
15
15
12
5.0
4.5-5.0
3.5-4.0
1.7-2.5
1.7-2.5
0.8-1.2
0.8-1.2
100-6000
32
1.0-1.2
8-10
4-8
4-8
1ml
Related Products

Concentration, Amount,
Loading, Recommended Number of Agarose, PAGE,
Applications
g/l
g
g(l)/lane
range,bp
fragments
%
%
TopVision Agaroses
Loading Dyes
50X TAE Buffer
10X TBE Buffer
p.484
p.428
p.430
p.430
p.476
Description
ORangeRuler DNA ladders are designed

for precise sizing of PCR products and other
double-stranded DNA fragments in agarose
or non-denaturing polyacrylamide gels. These
step ladders consist of ligated blunt end basic
unit repeats of 10, 15, 20, 50, 100, 200 or
500bp. They are not recommended for DNA
quantification.
ORangeRuler DNA ladders are supplied with
6X Orange DNA Loading Dye for sample DNA.
Orange G dye (migrates at 50bp in a 1%agarose gel) and can be used to monitor DNA
migration in agarose or polyacrylamide gels. It
is the preferred dye when visualization of small
DNA fragments is important.

10%glycerol.
Features

for 6 months).
Step ladders of 5, 10, 20, 50, 100, 200 or

500bp increments.
Sharp bands
Easy-to-remember fragment sizes.
Evenly spaced reference bands.
Readytouse premixed with 6X Orange
Loading Dye.

60mM EDTA.
Quality Control
Tested in appropriate gel electrophoresis applications. The DNA concentration of the complete
ladder determined spectrophotometrically. The
absence of nucleases confirmed by appropriate
tests.
Storage

electrophoresis
electrophoresis
electrophoresis
for electrophoresis
www.fermentas.com
422
p.431
p.431
p.432
p.433
p.433
ORangeRuler 5bp
DNA Ladder, readytouse
ORangeRuler 10bp
ORangeRuler 20bp
ORangeRuler 50bp
#SM1303
#SM1313
#SM1323
#SM0613
bp
150
100
90
80
70
60
50
40
30
10
25
20
1g/lane,
10cm length gel,
1X TBE, 5 V/cm, 1 h
15
30
100
90
80
70
60
50
20
40
30
10
20
1g/lane,
10cm length gel,
1X TBE, 5 V/cm, 1 h
10
50
45
40
35
150
10%polyacrylamide
50
45
40
35
30
25
20
15
100
10%polyacrylamide
100
300
200
180
160
140
120
100
80
40
40
20
20
1g/lane,
10cm length gel,
1X TBE, 5 V/cm, 1 h
10
1000
1000
500
450
400
350
500
450
400
350
300
250
60
60
0.5g/lane,
20cm length gel,
1X TBE, 8 V/cm, 3 h
0.5g/lane,
20cm length gel,
1X TBE, 8 V/cm, 3 h
300
200
180
160
140
120
100
80
bp
bp
300
250
200
200
150
150
100
50
0.25g/lane,
20cm length gel,
1X TBE, 5 V/cm, 1.5 h
5%polyacrylamide
bp
bp
bp
bp
10%polyacrylamide
bp
100
50
0.25g/lane,
20cm length gel,
1X TAE, 8 V/cm, 3 h
0.5g/lane,
20cm length gel,
1X TBE, 8 V/cm, 3 h
ORangeRuler 100bp
ORangeRuler 200bp
ORangeRuler 500bp
ORangeRuler 100+500bp
#SM0623
#SM0633
#SM0643
#SM0653
bp
bp
bp
bp
bp
1500
500
400
300
200
200
100
100
300
5%polyacrylamide
400
0.25g/lane,
20cm length gel,
1X TBE, 5 V/cm, 1.5 h
3000
2800
2600
2400
2200
2000
1800
1600
1400
1200
1000
800
600
400
200
0.25g/lane,
20cm length gel,
1X TAE, 7 V/cm, 1 h
6000
5500
5000
4500
4000
3500
3000
2500
1000
900
800
700
600
500
2000
1500
1000
500
0.25g/lane,
20cm length gel,
1X TAE, 7 V/cm, 1 h
6000
5500
5000
4500
4000
3500
3000
2500
1000
900
800
700
600
1500
2000
1500
1400
1300
1200
1100
1000
900
800
700
600
500
400
300
200
100
0.25g/lane,
20cm length gel,
1X TAE, 7 V/cm,1 h
0.25g/lane,
20cm length gel,
1X TAE, 8 V/cm, 3 h
423
Conventional Lambda DNA Markers (15-48502bp)

DNA marker, marker #
Lambda DNA/EcoRI Marker, 1
Lambda DNA/HindIII Marker, 2
Lambda DNA/HindIII Marker, 2, readytouse
Lambda DNA/EcoRI+HindIII Marker, 3
Lambda DNA/EcoRI+HindIII Marker, 3,

readytouse
Lambda pUC MIx Marker, 4
Lambda DNA/Eco47I (AvaII) Marker, 13
Lambda DNA/Eco91I (BstEII) Marker, 15
Lambda DNA/Eco130I (StyI) Marker, 16
Lambda Mix Marker, 19
Lambda DNA/PstI Marker, 24
Supplied with:
6X DNA Loading Dye
Amount,
g
SM0281
SM0101.
SM0102
SM0103
SM0191.
SM0192
0.5
250 (5x50)
250 (5x50).
1250 (25x50)
250 (5x50)
250 (5x50).
1250 (25x50)
500
500.
2500
500
500.
2500
0.5 (1)
SM0193
0.1
250 (5x50)
500
0.5 (5)
SM0291
SM1051
SM0111
SM0161
SM0231
SM0361
0.5
0.5
0.5
0.5
0.5
0.5
50
250 (5x50)
250 (5x50)
250 (5x50)
250 (5x50)
250 (5x50)
100
500
500
500
500
500
0.5 (1)
0.5 (1)
0.5 (1)
0.5 (1)
0.5 (1)
0.5 (1)
0.5
0.1
0.5
0.5g/lane g(l)/lane
0.5 (1)
Range,
bp
Number of Agarose,
fragments
%
3530-21226
0.7
125-23130
1.0
125-21226
13
1.0
74-19329
23-8126
117-8453
74-19329
1503-48502
15-11501
6
36
14
11
14
29
1.0
1.5
1.0
1.0
0.7
1.5
0.5 (5)
0.5 (1)
2ml / 10ml
Description
Related Products

Concentration,
g/l
Cat. #
TopVision Agaroses
Loading Dyes
50X TAE Buffer
10X TBE Buffer
Klenow Fragment
Klenow Fragment, exo
dNTP Set
Modified dNTPs
ATP
0.5 M EDTA, pH8.0
p.484
p.428
p.430
p.430
p.250
p.251
p.466
p.470
p.244
p.469
p.477
p.476
Conventional Lambda DNA markers are used for

sizing of linear double-stranded large DNA fragments in agarose gels. Lambda DNA digested
to completion with the appropriate restriction
enzyme(s), purified and dissolved in storage
buffer. DNA fragments contain sticky ends.
Lambda DNA/HindIII Marker and Lambda
DNA/EcoRI+HindIII Marker are available in
readytouse versions premixed with 6X DNA
Loading Dye for direct loading (after heating)
onto agarose gels.
Markers supplied in TE buffer can be labeled
radioactively with T4 Polynucleotide Kinase
(#EK0031). Alternatively, they can be labeled
radioactively or non-radioactively with Klenow
Fragment (#EP0051) or Klenow Fragment,
exo (#EP0421) by the fill-in reaction (with the
exception of Lambda DNA/PstI Marker).
Features
Sizing and approximate quantification of large
DNA fragments.
Sharp bands.
Readytouse versions are premixed with 6X
DNA Loading Dye.


(for readytouse markers)
6X DNA Loading Dye

60mM EDTA.
Quality Control
marker determined spectrophotometrically. The
tests.
Storage
Store at -20C.

electrophoresis
electrophoresis
electrophoresis
for electrophoresis
7.1.4. DNA 3-end labeling by fill-in of
5-overhangs
www.fermentas.com
424
p.431
p.431
p.433
p.433
p.380
p.380
Lambda DNA/EcoRI Marker, 1
Lambda DNA/HindIII Marker, 2
Lambda DNA/EcoRI+HindIII Marker, 3
#SM0281
#SM0101/2/3
#SM0191/2/3
bp ng/0.5g
bp ng/0.5g
bp ng/0.5g
%
21226* 218.8 43.8
21226* 218.8 43.8
5804
5643
59.8 12.0
58.2 11.6
4878
50.3 10.1
3530* 36.4
7.3
2322
2027
23.9
20.9
4.8
4.2
564
5.8
1.2
125
1.3
0.3
76.5 15.3
23130* 238.4 47.7

9416 97.1 19.4
6557 67.6 13.5
4361* 45.0 9.0
7421
5148
4973
4268
3530*
53.1 10.6
51.3 10.3
44.0 8.8
36.4 7.3
2027
1904
1584
1375
20.9
19.6
16.3
14.2
4.2
3.9
3.3
2.8
947
831
9.8 1.95
8.6 1.7
564
5.8
1.2

0.5g/lane, 20cm length gel, 1X TAE buffer, 3 V/cm,

18 h (until bromophenol blue dye reached the bottom
of the gel)

Lambda pUC Mix Marker, 4
Lambda DNA/Eco47I (AvaII) Marker, 13
Lambda DNA/Eco91I (BstEII) Marker, 15
#SM0291
#SM1051
#SM0111
19329* 181.5 36.3

7743 72.5 14.5
5526 52.0 10.4
4254* 40.0 8.0
3280 45.5 9.1
2690 25.0 5.0
2322 21.5 4.3
1882 17.5 3.5
1489
14.0
2.8
1150
925
11.0
8.5
2.2
1.7
697
6.5
1.3
421
4.0
0.8

bp ng/0.5g
%
8126 83.8 16.8
6555 67.6 13.5
6442 66.4 13.3
3676 37.9 7.6
2606 26.9 5.4
2555 26.3 5.3
2134 22.0 4.4
2005 20.7 4.1
1951 20.1 4.0
1611* 16.6 3.3
1420 14.6 2.9
1284 13.2 2.6
985
10.2 2.0
974
10.0 2.0
894
9.2 1.8
597
6.2 1.2
590
6.1 1.2
513
5.3 1.1
511
5.3 1.1
433
4.5 0.9
398
4.1 0.8
345
3.6 0.7
310
3.2 0.6
308
3.2 0.6
0.5g/lane, 8cm length gel, 1X TAE, 7 V/cm, 1 h
bp ng/0.5g
bp ng/0.5g
125bp fragment is not visible and it comprises 0.3%.**
8453*
7242
6369
5687*
4822
4324
3675
87.1
74.7
65.7
58.6
49.7
44.6
37.9
17.4
14.9
13.1
11.7
9.9
8.9
7.6
2323
1929
23.9
19.9
4.8
4.0
1371
1264
14.1
13.0
2.8
2.6
702
7.2
1.4

272, 242, 215, 151, 88, 73, 67, 45, 42, 32, 29 *, 23bp
fragments are not visible and they comprise 2.7%.**
Lambda DNA/Eco130I (StyI) Marker, 16
Lambda Mix Marker, 19
Lambda DNA/PstI Marker, 24
#SM0161
#SM0231
#SM0361
bp ng/0.5g
1882
19.4
3.9
1489
15.3
3.1
925
421
9.5
4.3
1.9
0.9
19329* 199.3 39.9

7743 79.8 16.0
6223 64.2 12.8
4254* 43.9 8.8
3472 35.8 7.2
2690 27.7 5.5
48502
38416
33498
29946
24508
23994
19397*
17053
15004
61.5
69.5
60.5
54.0
44.5
43.5
35.0
31.0
27.0
12.3
13.9
12.1
10.8
8.9
8.7
7.0
6.2
5.4
12220 22.0
4.4
10086 18.0
8614* 15.5
8271 15.0
3.6
3.1
3.0
bp ng/0.5g
%
11501* 118.6 23.7
5077 52.3 10.5
4749 49.0 9.8
4507 46.5 9.3
2838 29.3 5.9
2556* 26.3 5.3
2459 25.3 5.1
2443 25.2 5.0
2140 22.1 4.4
1986 20.5 4.1
1700 17.5 3.5
bp ng/0.5g
224, 117bp fragments are not visible and they comprise 0.7%.**

0.5g/lane, 20cm length gel, 1X TAE buffer, 3 V/cm, 18 h.

(until bromophenol blue dye reached the bottom of the gel)
1159
1093
11.9
11.3
2.4
2.3
805
8.3
1.7
514
468
448
5.3
4.8
4.6
1.1
1.0
0.9
339
3.5 0.7
264
2.7 0.5
247
2.5 0.5
0.5g/lane, 8cm length gel,1X TAE, 7 V/cm,1 h
216, 211, 200, 164, 150, 94, 87, 72, 15bp fragments are
not visible and they comprise 2.3%.**
* the cohesive ends (the 12 nt cos site of lambda DNA) of fragments may anneal and form additional bands. These fragments can be separated by heating at 65C for 5min and then cooling on
ice for 3min.
** the shortest fragments (oblique) are not visible in standard electrophoresis. Fragment lengths are calculated using respective DNA sequence (see pp.411-412).
425
Conventional Phage and Plasmid DNA Markers (8-1353bp)

DNA marker, marker #
pBR322 DNA/BsuRI (HaeIII) Marker, 5
pUC Mix Marker, 8
pUC Mix Marker, 8, readytouse
FX174 DNA /BsuRI (HaeIII) Marker, 9
Concentration,
g/l
Amount,
g
SM0271
SM0301.
SM0302
SM0303
SM0251.
SM0252
0.5
50
50.
250 (5x50)
50
50.
250 (5x50)
100
100.
500
100
100.
500
Cat. #
0.1
0.5
0.5 (1)
0.5 (1)
0.1
50
100
0.5 (5)
SM0261
SM0121
0.5
0.5
50
100
0.5 (1)
0.5 (1)
SM0123
0.1
50
100
pUC19 DNA/MspI (HpaII) Marker, 23
SM0221.
SM0222
0.5
50.
250 (5x50)
100.
500
0.5 (1)
pUC19 DNA/MspI (HpaII) Marker, 23,

readytouse
SM0223
0.1
50
100
0.5 (5)
TopVision Agaroses
Loading Dyes
50X TAE Buffer
10X TBE Buffer
Klenow Fragment
Klenow Fragment, exo
dNTP Set
Modified dNTPs
ATP
0.5 M EDTA, pH8.0
p.484
p.428
p.430
p.430
p.250
p.251
p.466
p.470
p.244
p.469
p.477
p.476
www.fermentas.com
8-587
22
2.5
19-1118
17
1.7
72-1353
11
1.7
24-726
21
2.5
11-908
17
1.7
26-501
13
1.7
5.0
5.0
0.5 (5)
2ml / 10ml
Related Products

Number of Agarose, PAGE,

fragments
%
%
0.5 (1)
SM0253
Supplied with:
6X DNA Loading Dye
Range,
bp
0.5 (5)
FX174 DNA /BsuRI (HaeIII) Marker, 9,

readytouse
FX174 DNA /HinfI Marker, 10
pBR322 DNA/AluI Marker, 20
pBR322 DNA/AluI Marker, 20,
readytouse

electrophoresis
electrophoresis
electrophoresis
for electrophoresis
7.1.4. DNA 3-end labeling by fill-in of
5-overhangs
426
0.5
0.5g/lane g(l)/lane
Description
Features
Conventional Phage and Plasmid DNA markers

are classical molecular weight standards used
for sizing and approximate quantification of
linear double-stranded DNA fragments in agarose and non-denaturing polyacrylamide gels.
The markers are composed of phage or plasmid
DNA digested to completion with the appropriate restriction enzyme, purified and dissolved in
storage buffer. The DNA fragments contain blunt
or sticky ends, depending on the restriction
enzyme used for the markers preparation.
pUC Mix, X174 DNA/BsuRI, pBR322 DNA/
AluI and pUC19 DNA/MspI markers are available in readytouse versions premixed with
6X DNA Loading Dye for direct loading onto
agarose and polyacrylamide gels.
All conventional versions (supplied in storage
(TE) buffer) can be labeled radioactively with T4
Polynucleotide Kinase (#EK0031). Alternatively
pUC Mix, X174 DNA/HinfI and pUC19 DNA/
MspI markers can be labeled radioactively
or non-radioactively with Klenow Fragment
(#EP0051) or Klenow Fragment, exo (#EP0421)
using the fill-in reaction.
Sizing and approximate quantification of DNA.

Sharp bands.
Readytouse versions are premixed with 6X
DNA Loading Dye for direct loading and room
temperature storage.


(for readytouse markers)
6X DNA Loading Dye

60mM EDTA.
Quality Control
marker determined spectrophotometrically. The
tests.
Storage
p.431
p.431
p.432
Store at -20C.
p.433
p.433
p.380
p.380
pBR322 DNA/BsuRI (HaeIII) Marker, 5
pUC Mix Marker, 8
#SM0271
#SM0301/2/3
267
234
213
192
184
5%polyacrylamide

1X TBE, 5 V/cm, 1.5 h
587*
540*
502
458*
434
124
123
104
89
80
67
30.05
23.6
18.25
13.8
13.65
6.0
4.7
3.7
2.8
2.7
8.3
1.7

1X TBE, 5 V/cm, 1.5 h
64
57
51
404
1353 125.6 25.1

1078 100.1 20.0
872
81.0 16.2
331
603
56.0 11.2
310
281
271
234
194
28.8
26.1
25.2
21.7
18.0
5.8
5.2
5.0
4.3
3.6
118
72
11.0
6.7
2.2
1.3
242
190
147
111
110
67
bp
1353
1078
872
603
310*
281*
271*
234
194
118

1X TBE, 5 V/cm, 1.5 h
34, 34
1X TAE, 8 V/cm, 3 h

1X TAE, 8 V/cm, 3 h
21, 18, 11, 8bp fragments are not visible and comprise 5.4%.**
692
501*
489*
1118 68.75 13.8

881
54.2 10.8
692 42.55 8.5
501
62.2 12.4
489
60.7 12.1
404 50.15 10.0
331
41.1 8.2
242
190
147
111
110
bp ng/0.5g
bp
1118
881
5%polyacrylamide
13.5
12.4
11.5
10.5
10.0
6.1
5.4
4.9
4.4
4.2
2.8
2.8
2.4
2.0
1.8
67.3
61.9
57.6
52.5
49.8
30.6
26.8
24.4
22.0
21.1
14.2
14.1
11.9
10.2
9.2
#SM0251/2/3
bp ng/0.5g
bp
5%polyacrylamide
587
540
502
458
434
267
234
213
192
184
124
123
104
89
80
bp ng/0.5g
X174 DNA/BsuRI (HaeIII) Marker, 9
72

1X TAE, 8 V/cm, 3 h
26, 19bp fragments are not visible and comprise 0.9%.**
X174 DNA/HinfI Marker, 10
pBR322 DNA/AluI Marker, 20
pUC19 DNA/MspI (HpaII) Marker, 23
#SM0261
#SM0121/3
#SM0221/2/3
311
249
200
151
140
118
100
82
bp ng/0.5g
908
659
656
521
104.1
75.6
75.2
59.7
20.8
15.1
15.0
11.9
403
46.2
9.2
281
257
226
32.2
29.5
25.9
6.4
5.9
5.2
100
11.5
2.3
90
10.3
2.1

1X TAE, 8 V/cm, 3 h
40, 24bp fragment is not visible and comprise 1.9%.**
bp
501*
489*
404
501
489
404
331
93.3
91.0
75.2
61.6
18.7
18.2
15.0
12.3
242
190
147
111
110
67
34
34
45.0
35.4
27.4
20.7
20.5
12.5
6.5
6.5
9.0
7.1
5.5
4.1
4.1
2.5
6.3
6.3

1X TBE, 5V/cm, 1.5h

1X TBE, 5V/cm, 1.5h
66, 66
48
42
63, 57, 49, 46, 19, 15, 11bp fragments are not visible and
comprise 5.9%.**
242
190
147
111
110
67
34, 34
1X TAE, 8V/cm, 3h
Note
Loading the marker in 5% polyacrylamide gels is not recommended due to anomalous migration of 659, 656, 403
and 257 fragments.
331
5%polyacrylamide

1X TBE, 5 V/cm, 1.5 h
726
713
553*
500
427*
417*
413*
726
67.4 13.5
713
66.2 13.2
553
51.3 10.3
500
46.4 9.3
427
39.6 7.9
417
38.7 7.7
413
38.3 7.7
311
28.9 5.8
249
23.1 4.6
200
18.6 3.7
151
14.0 2.8
140
13.0 2.6
118
11.0 2.2
100
9.3 1.9
82
7.6 1.5
66, 66 6.1, 6.1 1.2,1.2
48
4.5 0.9
bp ng/0.5g
bp
5%polyacrylamide
bp ng/0.5g
26bp fragment is not visible and comprises 1.0%.**
* bands form an anomalous pattern on polyacrylamide gels.

** the shortest fragments (oblique) are not visible in standard electrophoresis. Fragment lengths are calculated using respective DNA sequence (see pp.411-412).
427
Loading Dyes
Related Products
TopVision Agaroses
50X TAE Buffer
10X TBE Buffer
DNA Markers/Ladders
p.484
p.430
p.430
pp.413-426
Description
Quality Control
DNA Loading dye solutions are used to prepare

DNA markers and DNA samples for loading on
agarose or polyacrylamide gels. The optimized
solutions contain different dyes (bromophenol
blue, xylene cyanol FF or orange G) for visual
tracking of DNA migration during electrophoresis. The presence of glycerol ensures that the
DNA forms a layer at the bottom of the well. The
EDTA included in the solutions binds divalent
metal ions and inhibits metal-dependent
nucleases.
6X DNA Loading Dye & SDS Solution is
recommended for agarose gel analysis of DNA
samples that contain high amounts of DNA
binding proteins. The presence of SDS eliminates
DNA-protein interactions and prevents gel-shifts.
2X RNA Loading Dye is mainly used for preparation of RNA samples for electrophoresis, but is
also used for denaturing DNA electrophoresis
since it contains the denaturing agent formamide.
Tested for DNA sample preparation prior to agarose gel electrophoresis. The absence of deoxyribonucleases confirmed by appropriate tests.
2X RNA Loading Dye tested for RNA sample
preparation prior to agarose gel electrophoresis.
The absence of ribonucleases confirmed by
appropriate tests.
100kb
Storage
for 12 months).
Bromphenol blue in TBE buffer

Bromphenol blue in TAE buffer
10kb
Xylene cyanol FF in TBE buffer

Xylene cyanol FF in TAE buffer
1kb
100bp
10bp
0.5
1.5
2.5
3 3.5
Agarose, %
4.5
5.5
Figure 9.3. Tracking dye migration in agarose gels.

electrophoresis
for electrophoresis
www.fermentas.com
428
p.433
p.433
Loading dye
6X DNA Loading
Dye
6X MassRuler
DNA Loading Dye
6X Orange DNA
Loading Dye
6X TriTrack DNA
Loading Dye
Cat. #
R0611
R0621
R0631
R1161
6X DNA Loading
R1151
Dye & SDS Solution
2X RNA Loading
Dye
R0641
Size, Composition
ml
Features
Applications
Migration of dyes
(1%agarose,
TAE or TBE buffers)
5x1
6X Solution
Two-color tracking of
10mM Tris
DNA migration during
0.03%bromophenol blue
electrophoresis.
0.03%xylene cyanol FF No DNA masking during gel
60%glycerol
visualization in UV light.
60mM EDTA (pH7.6
EDTA inhibits metal
adjusted with NaOH)
dependent nucleases.
5x1
One-color tracking of
6X Solution
10mM Tris
electrophoresis.
No DNA masking during gel
60%glycerol
60mM EDTA (pH7.6,
EDTA inhibits metal
adjusted with NaOH)
Analysis of large
DNA molecules.
Bromophenol blue:
Preparation of
TAE: 370bp
DNA for loading
TBE: 220bp
on agarose or
polyacrylamide gels.
5x1
6X Solution
10mM Tris
0.15%orange G
0.03%xylene cyanol FF
60%glycerol
60mM EDTA (pH7.6,
adjusted with NaOH)
Xylene cyanol FF:

Analysis of small
TAE: 4160bp
DNA molecules.
TBE: 3030bp
Preparation of
DNA for loading
on agarose or
Orange G:
TAE/TBE: <50bp
5x1
6X Solution
Three-color tracking of
10mM Tris
electrophoresis.
0.15%orange G
60%glycerol
EDTA inhibits metal
60mM EDTA (pH7.6,
adjusted with NaOH)
5x1

6X Solution
60%glycerol
1%SDS
100mM EDTA (pH7.6,

adjusted with Tris)
Two-color tracking of
electrophoresis.
EDTA inhibits metal
Preparation of
DNA for loading
on agarose or
Picture of
tracking
dyes*
Xylene cyanol FF:

TAE: 4160bp
TBE: 3030bp
Bromophenol blue:
TAE: 370bp
TBE: 220bp
Xylene cyanol FF:

TAE: 4160bp
Preparation of
TBE: 3030bp
DNA for loading
Bromophenol blue:
on agarose or
TAE: 370bp
polyacrylamide gels. TBE: 220bp
Orange G:
TAE/TBE: <50bp
Analysis of DNA
1% SDS eliminates DNAsamples with high
protein interactions, prevents
amounts of DNA
binding proteins.
appearance of additional
Kinetic experiments.
bands due to annealing
of DNA molecules with
DNA agarose gel
analysis after DNA
cohesive ends.
EDTA inhibits metalrestriction digestions,
ligation or dephosphorylation reactions.
Two-color tracking of DNA,

RNA migration during
Preparation of
2X Solution
95%formamide
electrophoresis.
DNA for loading on
denaturing gels
0.025%SDS
No DNA, RNA masking
during gel visualization in UV Preparation of
light.
RNA for loading on
0.025%ethidium bromide Denaturation of DNA, RNA
agarose or poly 0.5mM EDTA
due to the presence of
acrylamide gels.
formamide.
Xylene cyanol FF:

TAE: 4160bp
TBE: 3030bp
Bromophenol blue:
TAE: 370bp
TBE: 220bp
Xylene cyanol FF:

TAE: 4160bp
TBE: 3030bp
Bromophenol blue:
TAE: 370bp
TBE: 220bp
* for more detailed information regarding the migration rates of dyes in agarose and polyacrylamide gels see Tables 9.1 and 9.2 on p.431.
429
TopVision Agarose
Agarose
Cat. #
TopVision Low Melting Point Agarose
Applications
R0492
100 g Analytical electrophoresis of nucleic acids.

Preparative electrophoresis.
500 g Blotting assays.
R0801
25 g
R0491
TopVision Agarose
Size
Page
In-gel enzymatic processing experiments.

Analytical electrophoresis of nucleic acids.
Preparative electrophoresis using Agarase (#EO0461).
484
484
Related Products
50X TAE Buffer

10X TBE Buffer
Agarase
DNA Markers/Ladders
Loading Dyes
GeneJET Gel Extraction Kit
Silica Bead DNA Gel Extraction Kit
p.430
p.430
p.354
pp.413-426
p.428
p.349
p.353

electrophoresis
p.431
9.2. Preparation of agarose gels for DNA electrophoresis
p.431
Electrophoresis Buffers
Buffer
Cat. #
50X TAE Buffer

(Tris-acetate-EDTA)
10X TBE Buffer

(Tris-borate-EDTA)
B49
B52
Size 1X composition
1L
1L
Application and features
Usage recommendations
40mM Tris.
20mM acetic acid.
1mM EDTA.
pHof 50X TAE: 8.4
Use fresh 1X TAE both for the gel and for

Electrophoresis of nucleic acids in agarose and
the electrophoresis run.
To prepare 1X TAE buffer, add 20ml of
Used both as a running buffer and as a gel prepa50X TAE buffer to 980ml of deionized
ration buffer.
water and mix well.
Recommended for resolution of RNA and DNA
TAE buffer has a relatively low buffering
fragments larger than 1500 b(p), for genomic
capacity, therefore periodic replacement
DNA and for large supercoiled DNA.
of the buffer during prolonged electro Filtered through a 0.22 m membrane.
phoresis is recommended.
89mM Tris.
89mM boric acid.
2mM EDTA.
pHof 10X TBE: 8.3
Use fresh 1X TBE both for the gel and for

Electrophoresis of nucleic acids in agarose and
the electrophoresis run.
To prepare 1X TBE buffer, add 100ml of
Used both as a running buffer and as a gel prepa10X TBE buffer to 900ml of deionized
ration buffer.
water and mix well.
Recommended for electrophoresis of RNA and
Double-stranded linear nucleic acid
DNA fragments smaller than 1500 b(p).
molecules migrate about 10%slower in
Filtered through a 0.22 m membrane.
TBE buffer than in TAE buffer.
Related Products
Quality Control
Storage
TopVision Agaroses
DNA Markers/Ladders
Loading Dyes
The absence of endo-, exodeoxyribonucleases

and ribonucleases confirmed by appropriate
quality tests.
There are no time limitations for storage of the

electrophoresis buffers at room temperature.
If the buffer is stored at lower temperatures, a
precipitate may form, which is easily dissolved
by gentle heating.
p.484
pp.413-426
p.428

electrophoresis
electrophoresis
www.fermentas.com
430
p.431
p.431
p.432
9.1. General recommendations
for DNA electrophoresis
Choose optimal gel percentage according to
the tables below:
Table 9.1. Recommended agarose gels for electrophoretic separation of DNA fragments.
Agarose
gel,
%
Range of
efficient
separation,bp
Approximate positions of
tracking dyes,bp*
Bromophenol
blue
Xylene cyanol
FF
TBE
buffer
TAE
buffer
TBE
buffer
TAE
buffer
0.5
2000-50000
750
1150
13000
16700
0.6
1000-20000
540
850
8820
11600
0.7
800-12000
410
660
6400
8500
0.8
800-10000
320
530
4830
6500
0.9
600-10000
260
440
3770
5140
1.0
400-8000
220
370
3030
4160
1.2
300-7000
160
275
2070
2890
1.5
200-3000
110
190
1300
1840
1040
2.0
100-2000
65
120
710
3.0
25-1000
30
60
300
460
4.0
10-500
18
40
170
260
5.0
10-300
12
27
105
165
* Positions of the tracking dyes can only be estimated approximately

because the dye front migrates as wide band.
Table 9.2. Recommended polyacrylamide gels for

electrophoretic separation of DNA fragments (1).
Approximate positions of
tracking dyes*
Polyacrylamide gel
(with BIS at 1:20),
%(w/v)
Range of
efficient
separation
4.0
100-500 b
50 b
230 b
5.0
70-400 b
35 b
130 b
6.0
40-300 b
26 b
105 b
8.0
30-200 b
19 b
75 b
Bromophenol
blue
Xylene
cyanol FF
Denaturing gels
10.0
20-100 b
12 b
55 b
15.0
10-50 b
10 b
40 b
20.0
5-30 b
8b
28 b
1-10 b
6b
20 b
30.0
Non-denaturing gels
3.5
100-1000bp
100bp
460bp
5.0
80-500bp
65bp
260bp
160bp
8.0
60-400bp
45bp
12.0
50-200bp
20bp
70bp
15.0
25-150bp
15bp
60bp
20.0
5-100bp
12bp
45bp
* Positions of the tracking dyes can only be estimated approximately

because the dye front migrates as wide band.
Choose electrophoresis conditions according

to the recommendations below:
Size of the DNA
Voltage
< 1kb
5-10 V/cm
Buffer
TBE
1-5kb
4-10 V/cm
TAE or TBE
> 5kb
1-3 V/cm
TAE
Up to 10kb, fast
electrophoresis with
Express DNA ladders
up to 23 V/cm
TAE
Use the same DNA loading dye (supplied with

the DNA ladder/marker) for both sample DNA
and ladder/marker DNA.
If possible, load equal volumes of the sample
DNA and the ladder/marker DNA. The sample
can be diluted with 1X DNA loading dye.
Avoid high salt concentrations in the DNA

samples as this may cause band shift during
electrophoresis.
Following electrophoresis, visualize DNA by
gel staining in 0.5g/ml ethidium bromide
solution, SYBR Green I or GelRed.
9.1.1. Recommendations for accurate

in-gel DNA quantification
dNTPs, oligonucleotides, genomic DNA,
RNA, NTPs or buffer components can
interfere with spectrophotometrical measurements and may lead to inaccurate quantification of sample DNA. In these cases, it is
best to rely on gel quantification data.
For the most accurate quantification, use
video-densitometry analysis.
Use the same DNA loading dye solution (supplied with the DNA ladder/marker) for both the
sample DNA and the ladder/marker DNA.
Compare the sample band intensity with the
ladder band of the closest size.
If possible, adjust the concentration of the
sample to approximately equalize it with the
amount of DNA in the nearest band.
9.2. Preparation of agarose gels

for DNA electrophoresis
9.2.1. Non-denaturing agarose gel
electrophoresis
Use a flask of at least three times greater
volume than that of the solution to avoid
boiling over.
Use the same 1X electrophoresis buffer to
prepare the gel and to run electrophoresis.
Dilute 50X TAE Buffer (#B49) or 10X TBE
Buffer (#B52) to a 1X concentration immediately before use.
Use TBE buffer for analysis of DNA bands
smaller than 1500bp. For larger DNA, use
TAE buffer.
For intensified gel staining, add ethidium
bromide to both the gel and the electrophoresis buffer at a final 0.5g/ml concentration. Alternatively, stain the gel after
electrophoresis.
Wear gloves when handling ethidium bromide.
For reliable analysis of supercoiled/relaxed
plasmid DNA, ethidium bromide should not
be included in the electrophoresis buffer
or gel. The gel should be stained only after
electrophoresis is complete.
Ethidium bromide staining and exposure to
UV light may cause DNA alterations. Therefore,minimize UV exposure to a few seconds
if the purified DNA fragments will be used for
cloning.
Staining before electrophoresis with such

intercalating dyes as SYBR GreenI, GelRed
and others may cause abberant migration of
DNA bands and DNA Ladder, that may cause
mistakes in sizing of DNA. Perform DNA
staining step only after gel electrophoresis.
Preparation:
1. Weigh out the required amount of agarose
(#R0491 or #R0801) (depending on the gel
percentage) into an Erlenmeyer flask.
2. Add the appropriate volume of either 1X TBE
or 1X TAE buffer and swirl to mix.
3. Weigh the flask with the solution.
For high percentage gels (3-5%): add an
excess amount of distilled water to increase the
weight by 10-20%.
4. Boil the mixture in a microwave oven (at
medium power) until the agarose melts
completely; swirl the flask several times
while boiling. To prepare the highest quality
agarose gels of any percentage, an additional 3-5min of boiling after completely
melting the agarose is recommended. A significant amount of water evaporates during
this procedure and therefore restoring of the
initial weight (in step 5) is required to obtain
the desired percentage gel.
5. Weigh the flask again and if necessary, add
hot distilled water to restore the initial weight.
For high percentage gels (3-5%): check (by
weighing) that the excess 10-20%of water has
evaporated and, if needed, continue boiling to
remove any excess, or add hot distilled water to
restore the initial weight.
Optional: add ethidium bromide to a final
concentration of 0.5g/ml. Mix well and heat for
1min without boiling.
6. Cool the solution to 65-70C. Pour carefully
on a clean casting plate with an appropriate
comb. If bubbles appear, push them carefully away to the sides with a pipette tip.
7. Solidify the gel for approximately 30min
before use. Low percentage low melting point
agarose gels need to be solidified at 4C.
8. Immerse the gel into the desired electrophoresis buffer. Load the samples onto the gel.
9. Run electrophoresis at 5-7 V/cm until the
bromophenol blue runs approximately 2/3 of
the way down the gel.
10. After electrophoresis the gel can be stained
by immersing it into a 0.5g/ml ethidium
bromide solution for 15-20min, stained with
GelRed, SYBR Green I or any other DNA
staining technique.
Warning. Hot agarose solution should be
handled very carefully.
431
9.2.2. Alkaline agarose gel

electrophoresis
Double stranded DNA ladders are not

recommended for denaturing electrophoresis as they may form an atypical pattern.
However, these discrepancies are normally
acceptable for analysis of cDNA or other
ssDNA in alkaline gels.
Use a flask of at least three times greater
volume than that of the solution to avoid
boiling over.
Wear gloves when handling ethidium bromide.
1. Weigh out the required amount of agarose
(depending on the gel percentage) into an
Erlenmeyer flask.
2. Add the appropriate volume of the buffer
(30mM NaCl, 2mM EDTA, pH7.5) and
swirl to mix.
3-7. Follow steps from protocol 9.2.1.
8. Immerse the gel for at least one hour
into the alkaline electrophoresis buffer
(30mMNaOH, 2mMEDTA).
9. Load the samples.
10. Run electrophoresis at 3 V/cm in alkaline
electrophoresis buffer (30mM NaOH, 2mM
EDTA) until the dye runs approximately 2/3
of the way down the gel.
After electrophoresis the gel should be
immersed for 30min in 100-300ml of
0.5M Tris-HCl buffer, pH7.5 and then
stained in a 0.5g/ml ethidium bromide
solution for 30min. If staining is not sufficient, the whole procedure can be repeated.
Warning. Hot agarose solution should be
handled very carefully.

9.3.1. Non-denaturing PAGE
Reagents:
Deionized water
10X TBE Buffer (#B52)
20%acrylamide/bisacrylamide solution
TEMED
10%(w/v) ammonium persulfate (APS) in water
0.5g/ml ethidium bromide solution
Denaturant free loading dye solution for
sample and ladder DNA
1. For a nondenaturing 5%polyacrylamide gel
solution of 40ml, mix the following:
4ml
20%acrylamide/bisacrylamide
10ml
deionized water
26ml
Caution: acrylamide is a neurotoxin; always

wear gloves, safety glasses, and a surgical
mask when working with acrylamide powder.
2. Vigorously agitate the solution for 1min by
magnetic stirring to ensure complete mixing.
3. Add 48 l of TEMED and swirl the flask to
ensure that the solution is thoroughly mixed.
www.fermentas.com
432
4. Immediately add 240 l of fresh 10%(w/v)

APS and mix thoroughly.
5. Pour the acrylamide between the gel plates
and insert the comb.
Clamp the comb in place at the top of the
gel to avoid separation of the gel from the
plates as the acrylamide polymerizes. Allow
the gel to polymerize for 30min.
Note
Polymerization begins as soon as APS is added to the mixture,

so all subsequent steps must be performed quickly.
6. Remove the comb and any bottom spacers

from the gel. Wash the gel plates to clean
any spilled acrylamide and be sure that the
spacers are properly seated and clean. Fill the
lower reservoir of the electrophoresis tank with
1X TBE buffer. Initially, place the gel into the
lower tank at an angle to avoid the formation
of air bubbles between the plates and the gel
bottom. Clamp the gel plates to the top of the
electrophoresis tank and fill the upper reservoir
with 1X TBE so that the wells are covered.
7. Pre-run and warm the gel for at least
30min at 5 V/cm (constant voltage).
8. Prepare the ladders for electrophoresis as
recommended in the product insert or in
Table 9.3 on p.433.
Load the recommended volume of the
ladder, premixed with the appropriate loading dye solution. Use the same loading dye
for the sample DNA.
9. Run the gel at 5 V/cm, taking care to avoid
excessive heating. Run the gel for the time indicated in the certificate of analysis of the ladder.
10. Stain the gel in a 0.5g/ml ethidium
bromide aqueous solution for about 30min.
Examine the gel under the UV light.
9.3.2. Denaturing polyacrylamide/

urea gel electrophoresis
Reagents:
Deionized water
20%acrylamide/bisacrylamide solution
TEMED
10%(w/v) ammonium persulfate (APS) in
water
0.5g/ml ethidium bromide solution
UREA
1. Prepare denaturing 10%polyacrylamide gel
solution of 40ml, mix the following:
20%acrylamide/bisacrylamide
UREA
deionized water
3. Add 40 l TEMED and swirl the flask to

ensure thorough mixing.
4. Immediately add 400 l of fresh 10%(w/v)
APS and mix thoroughly.
5. Pour the acrylamide between the gel plates
and insert the comb.
6. Clamp the comb in place at the top of the
gel to avoid separation of the gel from the
plates as the acrylamide polymerizes. Allow
the gel to polymerize for 30min.
Note
Polymerization begins as soon as APS is added to the mixture,

so all succeeding actions must be performed promptly.
Run the gel:

1. After polymerization is complete, remove the
comb and any bottom spacers from the gel.
Fill the lower reservoir of the electrophoresis
tank with 1X TBE buffer. Initially, place the
gel into the lower tank at an angle to avoid
the formation of air bubbles between the
plates and the gel bottom. Clamp the gel
plates to the top of the electrophoresis tank
and fill the upper reservoir with 1X TBE so
that the wells are covered.
2. Pre-run and warm the gel for at least
30min at 5 V/cm (constant voltage).
Note
Heat the gel (buffer) during the whole run at 60-70C.
3. Wash the wells with 1X TBE buffer to

remove UREA and gel pieces.
4. Load the samples.
5. Run the gel at 6 V/cm until the lower dye
front reaches the three thirds of the gel.
bp
bp
300
200
150
300
100
100
75
75
50
50
35
25
4ml
20
10ml
15
19.2 g (to 8 M final

concentration)
to 40ml
Caution: acrylamide is a neurotoxin; always

wear gloves, safety glasses, and a surgical
mask when working with acrylamide powder.
2. Vigorously agitate the solution by magnetic
stirring to ensure complete mixing and solving of UREA powder.
200
150
25
Figure 9.4. Migration of GeneRuler Ultra Low

Range and Low Range DNA ladders under dena
turing conditions.
Ladders are prepared for electrophoresis using formamide containing 2X RNA Loading Dye (#R0641) and
separated on 10%denaturing PAGE with 8 M UREA.
1 GeneRuler Ultra Low Range DNA Ladder (#SM1211)
2 GeneRuler Low Range DNA Ladder (#SM1191)
6. Soak the gel for about 15min in 1X TBE to
remove the urea prior to staining.
7. Stain the gel in a 0.5g/ml ethidium bromide aqueous solution for about 30min.
8. Examine the gel under the UV light.
Note
Double stranded DNA ladders may form an atypical pattern in

denaturing electrophoresis. However, GeneRuler Ultra Low
Range and Low Range DNA Ladders can be successfully used
for sizing ssDNA (see Fig.9.4).
1. Add 1 volume of 6X DNA Loading Dye &

SDS Solution to 5 volumes of DNA sample.
2. Mix well and heat at 65C for 10minutes.
3. Chill on ice, spin down and load.
Note
The prepared sample can be stored at -20C and reused for

electrophoresis after several freeze-thaw cycles.
9.4. Preparation of DNA samples

for electrophoresis
9.4.1. Preparation of DNA samples for
conventional DNA electrophoresis
6X DNA Loading Dye (#R0611), 6X MassRuler
DNA Loading Dye (#R0621), 6X Orange DNA
Loading Dye (#R0631), 6X TriTrack DNA
Loading Dye (#R1161) are all used according to
below protocol:
1. Add 1 volume of 6X DNA loading dye to
5volumes of DNA sample.
2. Mix well, spin down and load.
9.4.2. Preparation of DNA samples for

denaturing polyacrylamide/urea gel
electrophoresis
Note
Use the same loading dye solution for the sample and the
ladder DNA.
1. Mix the DNA sample with an equal volume

of 2X RNA Loading Dye (#R0641).
2. Heat at 95C for 5min.
3. Chill the sample on ice for 3min.
4. Keep samples on ice while loading.
9.4.3. Preparation of DNA samples

for denaturing alkaline agarose gel
electrophoresis
1. Mix 5 volumes of the DNA sample or ladder
with one volume of 6X alkaline electrophoresis
loading buffer (180mM NaOH, 6mMEDTA,
18%Ficoll 400, 0,05%bromcresol green).
2. Heat samples and ladder at 70C for 5min,
then chill on ice for 3minutes. Load onto
the gel.
9.4.4. Preparation of DNA samples

containing DNA binding proteins
Use 6X DNA Loading Dye & SDS Solution
(#R1151) to prevent the appearance of additional bands or gel shifts when analyzing:
probes after DNA restriction digestions, ligation or dephosphorylation reactions,
DNA samples with high amounts of DNA
binding proteins,
DNA molecules with cohesive ends,
or to stop an enzymatic reaction during
kinetic experiments.
Figure 9.5. The effect of SDS on electrophoresis

on resolving band shifts.
M GeneRuler DNA Ladder Mix (#SM0331)
1 0.5g DNA prepared for loading with 6X DNA
Loading Dye (#R0611)
2 0.5g DNA prepared for loading with 6X DNA
Loading Dye & SDS Solution (#R1151)
3 0.5g DNA digested with TsoI (#ER1991),
prepared for loading with 6X DNA Loading Dye
4 0.5g DNA digested with TsoI, prepared for loading with 6X DNA Loading Dye & SDS Solution
5 0.4g of the 2 DNA fragment ligation mixture
prior to ligation
after the ligation, prepared for loading with 6X
DNA Loading Dye
after the ligation, prepared for loading with 6X
DNA Loading Dye & SDS Solution
9.5. Preparation of DNA ladders/markers for electrophoresis

Table 9.3. Recommendations for loading of DNA ladders/markers supplied in TE buffer.
Conventional formulation (supplied in TE buffer) DNA ladders/markers
Technical specifications
Amount used per 1mM width of a
gel lane
Heating
GeneRuler DNA
ladders
Conventional Lambda DNA

markers
Conventional Phage &

Plasmid DNA markers
0.1g (0.2 l) for agarose gel

0.2g (0.4 l) for PAGE

0.2g (0.4 l) for PAGE
Do not heat
Heat at 65C for 5min;

chill on ice for 3min before use
Do not heat
1 l (0.5g).
2 l.
9 l
1 l (0.5g).
2 l.
9 l
1 l (0.5g).
2 l.
9 l
I. Loading on agarose gel:

DNA ladder/marker
loading dye
Water, nuclease-free (#R0581)
Mix gently and load on gel

II. Loading on polyacrylamide gel:
DNA ladder/marker
loading dye
Water, nuclease-free (#R0581)
2 l (1g).
0.5 l.
0.5 l
Not recommended for PAGE
2 l (1g).
0.5 l.
0.5 l
Mix gently and load on gel
9.6. Labeling of DNA ladders/markers

For protocols see chapter 7 Molecular Labeling & Detection on p.380.
433
9.7. Separation of Express DNA ladders in different electrophoresis conditions

Table 9.8. ZipRuler Express DNA Ladder (#SM1373) separation at various electrophoresis conditions.
Table 9.4. GeneRuler Express DNA Ladder

(#SM1551) separation at various electrophoresis
conditions.
Duration of 0.8%
1%
1.2%
1.5%
2%
2.5%
electro agarose agarose agarose agarose agarose agarose
phoresis
TAE TBE TAE TBE TAE TBE TAE TBE TAE TBE TAE TBE
23 V/cm
5min
10min
15min
20min
Table 9.5. MassRuler Express HR Forward and

Reverse DNA Ladder (#SM1243 and #SM1253)
separation at various electrophoresis conditions.
Duration of
0.7%
electro
agarose
phoresis
TAE TBE
7V/cm
10min
20min
30min
40min
50min
1%
agarose
TAE
TBE
1.3%
agarose
TAE
TBE
1.5%
agarose
TAE
TBE
Duration
of electro
phoresis
10 V/cm
0.8% agarose
TAE
TBE
1% agarose
TAE
TBE
1.2% agarose
1.5%agarose
1.7% agarose
TAE
TAE
TAE
TBE
TBE
TBE
2% agarose
TAE
TBE
Ladder
Ladder
Ladder
Ladder
Ladder
Ladder
1 2 Mix 1 2 Mix 1 2 Mix 1 2 Mix 1 2 Mix 1 2 Mix 1 2 Mix 1 2 Mix 1 2 Mix 1 2 Mix 1 2 Mix 1 2 Mix
5min
10min
15min
20min
25min
1 ZipRuler Express DNA Ladder 1.
2 ZipRuler Express DNA Ladder 2.
Mix Ladder 1 and Ladder 2 mixed at equal volumes.
Excellent separation of all bands.

Incomplete separation of the two closest bands.
No separation.
Lowest bands run off of gel (8cm).
Table 9.6. MassRuler Express LR Forward and

Reverse DNA Ladder (#SM1283 and #SM1293)
Duration of 0.7%
1%
1.3%
1.5%
2%
electro agarose agarose agarose agarose agarose
phoresis
TAE TBE TAE TBE TAE TBE TAE TBE TAE TBE
7 V/cm
10min
20min
30min
40min
50min
Table 9.7. MassRuler Express Forward and

Reverse DNA Ladder Mix (#SM1263 and #SM1273)
Duration of
0.7%
electro
agarose
phoresis
TAE TBE
7V/cm
10min
20min
30min
40min
50min
1%
agarose
TAE
TBE
1.3%
agarose
TAE
TBE
1.5%
agarose
TAE
TBE
Reference
1. Sambrook, J., et al., Molecular Cloning. A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., 12.89, 5.42, 2001.
www.fermentas.com
434
Troubleshooting Guide
DNA electrophoresis
problem
1
Low intensity
of all or some
DNA bands
2
Smeared DNA
bands
3
Atypical
banding
pattern
4
Curved DNA
bands
5
DNA remains
in the gel well
6
Incorrect
quantification
data
1.1
Insufficient
amount of
DNA loaded
2.1
DNA
degradation by
nucleases
3.1
l marker not
heated prior to
loading
4.1
Gel
incompletely
immersed in
buffer
5.1
Poorly-formed
gel wells
6.1
Different
loading condi
tions for ladder
& sample
1.2
Insufficient
or uneven
staining
2.2
Incorrect elec
trophoresis
conditions
3.2
Denatured
DNA
4.2
Low sample
volume
5.2
Excess DNA
loaded
6.2
Incorrect
ladder band
chosen for
quantification
1.3
DNA
run off of
the gel
2.3
Gel shift
effect
3.3
Different
loading condi
tions for ladder
& sample
4.3
Incorrect elec
trophoresis
conditions
5.3
Contamination
of the DNA
sample
6.3
Improper
quantification
method
1.4
DNA
diffusion in
the gel
2.4
Excess
DNA loaded
3.4
Incorrect elec
trophoresis
conditions
4.4
Bubbles or
physical par
ticles in wells
or in the gel
5.4
Gel shift
effect
6.4
Uneven or high
background
staining of the
gel
1.5
DNA masking
by tracking
dyes
2.5
High salt
concentration
in samples
3.5
DNA
staining before
electropho
resis
2.6
Poorly
formed gel
wells
3.6
Atypical migra
tion due to
DNA sequence
or structure
6.5
DNA masking
by tracking
dyes
3.7
Gel shift
effect
3.8
High salt
concentration
in samples
435
Table 9.9. Troubleshooting guide for DNA electrophoresis.
Problem
Possible cause and recommended solution

1.1. Insufficient amount of ladder was loaded.
Follow the recommendations for loading described in the certificate of analysis of the DNA ladders/markers
(~0.1-0.2g per 1mM gel lane width).
1.2. Insufficient or uneven staining.

Following electrophoresis, visualize DNA by staining in ethidium bromide solution (final concentration
0.5g/ml) or SYBR Green I.
Alternatively, if the DNA will not be used for cloning, add ethidium bromide to both the gel and electrophoresis buffer at a final 0.5g/ml concentration.
After alkaline agarose gel electrophoresis the gel should be immersed for 30min in 300ml 0.5 M Tris-HCl
buffer, pH7.5 and only later stained in a 0.5g/ml ethidium bromide solution for 30min.
After denaturing polyacrylamide gel electrophoresis with urea, soak the gel for about 15minutes in 1X TBE to
remove the urea prior to staining. Stain the gel in 0.5g/ml ethidium bromide in 1X TBE solution for 15min.
Make sure that the gel is immersed completely in the staining solution.
1. Low intensity of all or some of 1.3. DNA run off the gel.
Perform electrophoresis until the bromophenol blue dye passes 2/3 (orange G, 4/5) of the gel. Refer to the
the DNA bands
table on p.431 for migration of tracking dyes in different gels.
Make sure that the entire gel is immersed completely in the electrophoresis buffer during the run. Make sure
that gel and apparatus are positioned horizontally during the run.
1.4. DNA diffusion in the gel.
Avoid prolonged electrophoresis or excessive staining and destaining procedures as this may cause diffusion
of smaller DNA fragments in the gel.
Avoid long term storage of the gel before taking a picture, as this may cause diffusion of DNA fragments and
low band intensity.
1.5. DNA masking by electrophoresis tracking dyes.
Do not exceed the amount of electrophoresis tracking dyes used for sample/ladder preparation. Use the
loading dye solutions supplied with every Fermentas DNA ladder/marker, as these solutions contain amount
of tracking dyes which will not mask DNA under UV light.
Prepare DNA ladders and probes according to recommendations on p.433.
2.1. DNA degradation by nucleases.
Use fresh electrophoresis buffers, freshly poured gels, nuclease free vials and tips tominimize nuclease
contamination of DNA solutions.
2. Smeared DNA bands
2.2. Incorrect electrophoresis conditions.

Prepare gels according to the recommendations on p.431, always use the same electrophoresis buffer for both
the gel and running buffer.
Ensure that the whole gel is immersed completely in the electrophoresis buffer during the run.
Do not use an excessively high voltage for electrophoresis. Run the gels at 5-8 V/cm. To increase the band
sharpness, use a lower voltage for severalminutes at the beginning of electrophoresis. .
Excessively high voltage may result in gel heating and DNA denaturation.
For fast electrophoresis under high voltage (up to 23 V/cm) use GeneRuler or OGeneRuler Express DNA
ladders (#SM1551/2/3 or #SM1563, p.417).
An excessively low voltage during the entire run may result in diffusion of bands during electrophoresis.
To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value
(usually 5-8 V/cm, depending on the ladder) one has to:
measure the distance between electrodes (cathode and anode) X,cm
and multiply that X,cm value by the recommended voltage (Y, V/cm)
the result (X,cm x recommended Y, V/cm) is Z recommended voltage to be applied.
2.3. Gel shift effect.
DNA binding proteins, such as ligases, phosphatases or restriction enzymes may alter DNA migration on gels
and cause the DNA to remain in the gel well or gel shifting.
Lambda DNA or other DNA with long complementary overhangs may anneal and migrate atypically (see p.425).
To correct for the above mentioned effects, use 6X DNA Loading Dye & SDS Solution (#R1151, p.429) which is
supplemented with 1%SDS to eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends.
Always heat these samples with SDS at 65C for 10min, chill on ice, spin down and load.
www.fermentas.com
436
(continued on next page)
Problem

2.4. Excess DNA loaded.
(~0.1-0.2g per 1mM gel lane width) or in the Table 9.3 on p.433. Use similar DNA quantities for the samples.
2. Smeared DNA bands
2.5. High salt concentration in the sample.

Samples containing high concentrations of salts may result in smeared or shifted band patterns.
Ethanol precipitation and washing the pellet with ice cold 75%ethanol or spin column purification prior to
resuspending the sample in water or TE buffer helps to eliminate salts present in the sample.
2.6. Poorly formed (slanted) gel wells.
When inserting the comb into the gel, make sure that it is vertical to the gel surface and stable during gel
casting and solidification.
3.1. Lambda DNA marker was not heated prior to loading.
All DNA markers generated from Lambda DNA, as well as lambda DNA digestion products should be heated
at 65C for 5min and chilled on ice before loading on the gel in order to completely denature the cohesive
ends (the 12 nt cos site of lambda DNA) that may anneal and form additional bands. See p.433 for preparation
of lambda markers for electrophoresis.
3.2. Denatured DNA.
Excessively high voltage may result in gel heating and DNA denaturation.
To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (often
5-8 V/cm, depending on the ladder) one has to:
and multiply that X,cm value by the recommended voltage (Y, V/cm)
the result (X,cm x recommended Y, V/cm) is Z recommended voltage to be applied.
For non-denaturing electrophoresis use the loading dye solutions supplied with every Fermentas DNA ladder/
marker, as these solutions do not contain denaturing agents.
Prepare DNA ladders and probes according to recommendations on p.433.
Do not heat them before loading. Heating is required only for lambda DNA markers.
3. Atypical banding pattern
3.3. Different loading conditions for the sample and ladder DNA.
Always use the same loading dye solution (supplied with the DNA ladder/marker) for both the sample DNA
and the ladder/marker DNA.
If possible, always load equal or very similar volumes of the sample DNA and the ladder/marker DNA. The
sample can be diluted with 1X loading dye.
Excessive electrophoresis run times or voltage may result in migration of small DNA fragments off of the gel.
Very short or slow electrophoresis may result in incompletely resolved bands.
Run gels at 5-8 V/cm until the bromophenol blue passes 2/3 (orange G, 4/5) of the gel. Refer to the
Table9.1 on p.431 for migration of tracking dyes in different gels.
ladders (#SM1551/2/3 or #SM1563, p.417).
TAE buffer is recommended for analysis of DNA fragments larger than 1500bp and for supercoiled DNA.
TBE buffer is used for DNA fragments smaller than 1500bp and for denaturing polyacrylamide gel electrophoresis. Large DNA fragments will not separate well in TBE buffer.
The correct gel percentage is important for optimal separation of the ladder DNA; prepare gels according to
recommendations on p.431. When preparing agarose gels always adjust the volume of water to accommodate
for evaporation during boiling. Otherwise, the gel percentage will be too high and result in bad separation of
larger DNA bands.
Refer to the Table9.1 on p.431 for the range of effective separation of DNA in different gels.
437
Problem

3.5. DNA staining before electrophoresis using fluorescent dyes.
Ethidium bromide interferes with separation of large DNA fragments. Do not include ethidium bromide in the
gel and run buffer when large DNA (more than 20kb) or supercoiled DNA is analyzed. Stain the gel following
electrophoresis in a 0.5g/ml ethidium bromide solution for 30min.
Staining before electrophoresis with such intercalating dyes as SYBR GreenI, GelRed and others may cause
abberant migration of DNA bands and DNA Ladder, that may cause mistakes in sizing of DNA. Perform DNA
staining step only after gel electrophoresis.
3.6. Atypical migration due to differences in DNA sequence or structure.

During high resolution electrophoresis DNA fragments of equal size can migrate differently due to differences in DNA sequences. AT rich DNA may migrate slower than an equivalent size GC rich DNA fragment.
The sequences of Fermentas DNA ladders are chosen to allow for highly accurate DNA migration according
to size, however, due to differencies in nucleotide sequence or the overall DNA structure, sample migration
can sometimes slightly differ from ladder band migration.
DNA structures such as nicked, supercoiled or dimeric molecules will always show different mobility on gels
compared to an equivalent DNA size standard. See the picture below for migration of plasmid DNA forms:
bp
3. Atypical banding pattern
10000
8000
6000
5000
4000
3500
3000
2500
2000
1500
1000
750
500
250
1 GeneRuler 1kb DNA Ladder (#SM0311)

2 Undigested plasmid pUC19 2,7kb DNA, forms:
upper band (~4kb) dimeric plasmid
below, less visible (~3.5kb) nicked plasmid
lowest band (~1.9kb) supercoiled plasmid.
3 Linearized plasmid pUC19 (2,7kb) migrates according to its size
High level DNA modifications such as methylation, labeling with biotin or large fluorescent molecules also
result in slower migration compared to unmodified DNA of the same size.
The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes
may alter DNA migration in the gel or cause the DNA to remain in the gel wells.
Lambda DNA or other DNA with long complementary overhangs may anneal resulting in an atypical migration pattern.
To eliminate these effects, use 6X DNA Loading Dye & SDS Solution (#R1151) which is supplemented with
1%SDS to eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends.
High salt concentration in the sample may also cause gel shift effects, see below (3.8).
3.8. High salt concentration in the sample.

Samples with a high salt concentration may give smeared or shifted band patterns.
Ethanol precipitation and washing the pellet with ice cold 75%ethanol or spin column purification prior
resuspending DNA in water or TE buffer, helps eliminate salt from the sample.
www.fermentas.com
438
Problem

4.1. Gel incompletely immersed in electrophoresis buffer.
Electrophoresis buffer should completely cover the entire gel during sample loading and run.
4.2. Low sample volume.
The sample or the ladder volume should be large enough to fill 1/3 of the total capacity of the well. Large
wells should not be used with small sample volumes. If needed the sample volume can be adjusted with
1Xloading dye.
4. Curved DNA bands

Do not use an excessively high voltage for electrophoresis. Run the gels at 5-8 V/cm. Tominimize band curving, use a lower voltage for severalminutes at the beginning of electrophoresis.
ladders (#SM1551/2/3 or #SM1563, p.417).
To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (which
is in many cases 5-8 V/cm, depending on the ladder) one has to:
and multiply the X value by the recommended voltage (Y, V/cm)
the result (X x Y) is the recommended voltage to be applied.
4.4. Bubbles or physical particles in the gel wells or in the gel.
Use pure water, clean flasks and clean equipment for preparation of gels.
Pour the gel slowly avoiding formation of bubbles. Bubbles can be removed with a pipette tip.
5.1. Poorly formed gel wells.
Remove the gel comb only after complete polymerization of the gel. Pour the buffer onto the gel immediately.
Rinse the wells with electrophoresis buffer to remove urea from denaturing polyacrylamide gels prior to loading the sample.
5.2. Excess DNA loaded.
(~0.10.2g per 1mm gel lane width) or in the Table 9.3 on p.433. If possible load the same quantity of the sample.
5. DNA remains in the gel well
5.3. Contamination of the DNA sample.

Ensure that your sample DNA solution does not contain any precipitate.
The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes
may alter DNA migration in the gel and cause the DNA to remain in the gel wells.
Lambda DNA or other DNA with long complementary overhangs may anneal resulting in an atypical band
migration pattern.
To eliminate these effects, use 6X DNA Loading Dye & SDS Solution which is supplemented with 1%SDS to
eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends.
6. Incorrect quantification data
6.1. Different loading conditions for the sample and the ladder DNA.
Always use the same loading dye solution (supplied with the DNA ladder/marker) for both the sample DNA
and the ladder/marker DNA.
If necessary, adjust the concentration of the sample to approximately equalize it with the amount of DNA in
the nearest band.
Use equal or very similar volumes of the sample DNA and the ladder/marker DNA. The sample can be diluted
with 1X loading dye solution.
6.2. Incorrect ladder band chosen for quantification of the sample.
Always compare the sample band with a ladder band of similar size.
6.3. Improper quantification method used.
If possible, quantify by video-densitometry while subtracting the gel background as this method is more
precise than a visual comparison of the bands.
439
Problem
6. Incorrect quantification data

6.4. Uneven staining of the gel and high background staining can also interfere with gel quantifica
tion results.
Ensure that the gel is immersed completely in the staining solution.
Following electrophoresis, visualize DNA by staining in ethidium bromide solution (final concentration
0.5g/ml) or SYBR Green I. Do not exceed the recommended concentration of the dye for staining.
Avoid prolonged staining for more than 30min as this may result in high background.
If the gel is to be stained during the run, ensure that the ethidium bromide is included in both the gel and
running buffer, otherwise the staining will be uneven.
After alkaline agarose gel electrophoresis the gel should be immersed for 30min in 300ml of
0.5MTrisHCl buffer, pH7.5 and only later stained in a 0.5g/ml ethidium bromide solution for 30min.
After denaturing polyacrylamide gel electrophoresis with urea, soak the gel for about 15min in 1X TBE to
remove the urea prior to staining. Stain the gel in 0.5g/ml ethidium bromide in 1X TBE solution for 15min.
6.5. DNA masking by electrophoresis tracking dyes.

Do not exceed the recommended amount of electrophoresis tracking dyes used for sample/ladder preparation. Use the loading dye solutions supplied with every Fermentas DNA ladder/marker, as these solutions
contain equilibrated amount of tracking dyes which will not mask DNA under UV light.
Prepare DNA ladders and probes according to the recommendations on p.433.
www.fermentas.com
440

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All Fermentas products are

manufactured in class D clean room

NoLimits Individual DNA Fragments and Custom DNA Ladders............................. 413

45min-18 h Classical markers

For patent and license information see p.557

MassRuler Low Range DNA Ladder, #SM0383, p.419

MassRuler DNA Ladder Mix, #SM0403, p.419

FastRuler Ultra Low Range DNA Ladder, #SM1233, p.420

ORangeRuler 5bp DNA Ladder, #SM1303, p.423

ZipRuler Express DNA Ladder 1, #SM1373, p.421

pBR322 DNA/BsuRI Marker, 5, #SM0271, p.426

Bulk quantities and custom formulations available upon request

FastRuler Middle Range

FastRuler Low Range

FastRuler Ultra Low Range

MassRuler Express HR Forward

MassRuler High Range

MassRuler Express Forward

MassRuler Express LR Forward

GeneRuler High Range

GeneRuler 1kb DNA Ladder

GeneRuler DNA Ladder Mix

GeneRuler Express DNA Ladder

GeneRuler 100bp Plus DNA Ladder

GeneRuler 100bp DNA Ladder

GeneRuler 1kb Plus DNA Ladder

GeneRuler 50bp DNA Ladder

GeneRuler Low Range DNA Ladder

GeneRuler Ultra Low Range

MassRuler Low Range

Reference for DNA Ladders and Markers

For patent and license information see p.557

ZipRuler Express DNA

Bulk quantities and custom formulations available upon request

Lambda pUC Mix, 4

For patent and license information see p.557

Chromatography-purified individual DNA

Storage Buffer (TE buffer)

1.1%TopVision Agarose (#R0491), 1X TAE, 7 V/cm, 30min.

NoLimits Custom DNA Ladders Service

Bulk quantities and custom formulations available upon request

GeneRuler and OGeneRuler DNA Ladders (10-20000bp)

GeneRuler DNA ladders are mixtures of chromatography-purified individual DNA fragments.

Ideal for both DNA sizing and approximate

Storage Buffer (TE buffer)

GeneRuler Storage and Loading Buffer

6X DNA Loading Dye

OGeneRuler Storage and Loading Buffer

6X Orange DNA Loading Dye

Protocols and Recommendations

For patent and license information see p.557

GeneRuler 1kb DNA Ladder

GeneRuler 1kb Plus DNA Ladder

GeneRuler 1kb Plus DNA Ladder,

OGeneRuler 50bp DNA Ladder,

GeneRuler Ultra Low Range DNA

OGeneRuler Ultra Low Range DNA

OGeneRuler Low Range DNA

OGeneRuler Express DNA Ladder,

Bulk quantities and custom formulations available upon request

GeneRuler 1kb Plus DNA Ladder

1000 60.0 12.0

2000 20.0 4.0