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GeneRuler™ DNA Ladders

GeneRuler™ High Range DNA Ladder, 10,171-48,502 bp

Catalog#	Size, concentration	Supplied with:	Certificate of Analysis	MSDS
Catalog#	Size, concentration	6X DNA Loading Dye	Certificate of Analysis	MSDS
SM1351	50 µg (0.5 µg/µl)	1.00 ml	SM1351
SM1352	250 (5x50) µg (0.5 µg/µl)	2 x 1.00 ml	SM1352

Product information

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Features

Ideal for both DNA sizing and approximate quantification.
Ideal for fast sizing of high molecular weight DNA fragments
Sharp bands.
Supplied with loading dye for sample DNA.

Description

GeneRuler™ High Range DNA Ladder is recommended for fast sizing and approximate quantification of high molecular weight double-stranded DNA on agarose gels (in 1.5 hour, 0.4% agarose gel). The ladder is a mixture of chromatography-purified individual DNA fragments.

GeneRuler™ High Range DNA Ladder can be labeled radioactively with T4 Polynucleotide Kinase, see protocol.)

The ladder is supplied with 6X DNA Loading Dye for sample DNA.

Storage Buffer (TE buffer)

10 mM Tris-HCl (pH 7.6) and 1 mM EDTA.

6X DNA Loading Dye

10 mM Tris-HCl (pH 7.6), 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol and 60 mM EDTA.

Quality Control

Well-defined bands are formed during agarose gel electrophoresis.
The DNA concentration is determined spectrophotometrically.
The absence of nucleases is confirmed by a direct nuclease activity assay.

Storage

Store at -20°C.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

General Recommendations for DNA Electrophoresis

Use the same DNA loading dye (supplied with the DNA ladder/marker) for both the sample DNA and the ladder/marker DNA.
If possible, always 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 bands to shift during electrophoresis.
Following electrophoresis, visualize DNA by staining in 0.5 µg/ml ethidium bromide solution or SYBR® Green I.

Choose the gel percentage according to the tables below:
Table 1. Recommended Agarose Gels for Electrophoretic Separation of DNA Fragments.

Agarose gel, %	Range of effective 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
2.0	100-2000	65	120	710	1040
3.0	25-1000	30	60	300	460
4.0	10-500	18	40	170	260
5.0	10-300	12	27	105	165

Table 2. Recommended Polyacrylamide Gels for Electrophoretic Separation of DNA Fragments (1).

Polyacrylamide gel (with BIS at 1:20), % (w/v)	Range of effective separation*	Approximate positions of tracking dyes*
Polyacrylamide gel (with BIS at 1:20), % (w/v)	Range of effective separation*	Bromophenol blue	Xylene cyanol FF
Denaturing gels
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
10.0	20-100 b	12 b	55 b
15.0	10-50 b	10 b	0 b
20.0	5-30 b	8 b	28 b
30.0	1-10 b	6 b	20 b
Non-denaturing gels
3.5	100-1000 bp	100 bp	460 bp
5.0	80-500 bp	65 bp	260 bp
8.0	60-400 bp	45 bp	160 bp
12.0	50-200 bp	20 bp	70 bp
15.0	25-150 bp	15 bp	60 bp
20.0	5-100 bp	12 bp	45 bp

Note

* Positions of the tracking dyes can only be estimated approximately because the dye front migrates as wide band. The following guidelines are recommended:

Only high purity agarose should be used. TopVision™ Agarose was used to prepare the gels.
Only freshly prepared electrophoresis buffers should be used. The buffers were prepared from Fermentas 50X TAE Buffer and 10X TBE Buffer.

Choose electrophoresis conditions according to the recommendations below:

Size of the DNA Voltage Buffer

<1 kb 5-10 V/cm TBE

1-5 kb 4-10 V/cm TAE or TBE

> 5 kb 1-3 V/cm TAE

Up to 10 kb, fast electrophoresis with Express DNA ladders up to 23 V/cm TAE

Recommendations for Accurate Gel Quantification

Always use the same DNA loading dye (supplied with the DNA ladder/marker) for both the sample DNA and the ladder/marker DNA.
Always compare the sample band 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.
dNTPs, oligonucleotides, genomic DNA, RNA, NTPs or buffer components can interfere with spectrophotometrical measurements and 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.

Reference

Sambrook, J., et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 12.89, 5.42, 2001.

Preparation of DNA Ladders/Markers for Electrophoresis

Table 1. Recommendations for loading the conventional formulation (supplied in TE buffer) DNA ladders/markers.

Technical specifications	Conventional formulation (supplied in TE buffer) DNA ladders/markers
Technical specifications	GeneRuler™ DNA ladders	Lambda DNA markers	Phage & Plasmid DNA markers	Markers for Genomic DNA Analysis
Supplied amount / number of applic.	50 µg (100 µl) is sufficient for: 100 applic. on agarose gel 50 applic. on native PAGE	50 µg (100 µl) is sufficient for 100 applic. on agarose gel	50 µg (100 µl) is sufficient for: 100 applic. on agarose gel 50 applic. on native PAGE	6 µg (30 µl) is sufficient for 120 applic. on agarose gel
Amount used per 1 mm width of a gel lane	0.1 µg (0.2 µl) for agarose gel 0.2 µg (0.4 µl) for PAGE	0.1 µg (0.2 µl) for agarose gel	0.1 µg (0.2 µl) for agarose gel 0.2 µg (0.4 µl) for PAGE	6 ng
Dilution	Not needed	Not needed	Not needed	Mix 1 µl (0.2 µg) of DNA marker with 39 µl of nuclease-free water
Heating	Do not heat	Heat at 65°C for 5 min; chill on ice for 3 min before use	Do not heat	Heat at 65°C for 5 min; chill on ice for 3 min before use
I. Loading on agarose gel:
DNA ladder/marker loading dye Water, nuclease-free	1 µl (0.5 µg) 2 µl 9 µl	1 µl (0.5 µg) 2 µl 9 µl	1 µl (0.5 µg) 2 µl 9 µl	10 µl (50 ng) of diluted marker 1 µl
Mix gently and load on gel
II. Loading on polyacrylamide gel:
DNA ladder/marker loading dye Water, nuclease-free	2 µl (1 µg) 0.5 µl 0.5 µl	Not recommended for PAGE	2 µl (1 µg) 0.5 µl 0.5 µl	Not recommended for PAGE
Mix gently and load on gel

Table 2. Recommendations for loading ready-to-use DNA ladders/markers.

Technical specifications	DNA ladders/markers, ready-to-use
Technical specifications	GeneRuler™ & O'GeneRuler™ DNA ladders	MassRuler™ DNA ladders	FastRuler™ DNA ladders	O'RangeRuler™ DNA ladders	ZipRuler™ Express DNA ladders	Lambda DNA markers	Phage & Plasmid DNA markers
Supplied volume/number of applic.	500 µl for 100 applic.	2x500 µl for 50-200 applic.	2x500 µl for 50-333 applic.	500 µl for 100 applic.	2x500 µl for 100-200 applic.	500 µl for 100 applic.	500 µl for 100 applic.
Heating	Do not heat					Heat at 65°C for 5 min; chill on ice for 3 min before use	Do not heat
Mix gently and load on gel
Volume per 1 mm width of a gel lane	1-2 µl	variable	variable	1-2 µl	1-2 µl	1-2 µl	1-2 µl

Preparation of DNA Samples for Conventional DNA Electrophoresis

6X DNA Loading Dye, 6X MassRuler™ DNA Loading Dye, 6X Orange DNA Loading Dye, 6X TriTrack™ DNA Loading Dye are all used according to below protocol:

Add 1 volume of 6X DNA loading dye to 5 volumes of DNA sample.
Mix well, spin down and load.

Preparation of DNA Samples from Enzymatic Reaction Mixtures or with Samples Containing High Amounts of DNA Binding Proteins

Use 6X DNA Loading Dye & SDS Solution 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.

To mix 6X DNA Loading Dye & SDS Solution with sample DNA:

Add 1 volume of 6X DNA Loading Dye & SDS Solution to 5 volumes of DNA sample.
Mix well.
Heat at 65°C for 10 minutes.
Chill on ice, spin down and load. 0

Note

The prepared sample can be stored at -20°C and reused for electrophoresis after several freeze-thaw cycles.

Figure. The effect of SDS on electrophoresis of DNA samples containing high amounts of DNA binding proteins.
M – GeneRuler™ DNA Ladder Mix
1 – 0.5 µg lambda DNA prepared for loading with 6X DNA Loading Dye
2 – 0.5 µg lambda DNA prepared for loading with 6X DNA Loading Dye & SDS Solution
3 – 0.5 µg lambda DNA digested with TsoI, probe prepared for loading with 6X DNA Loading Dye
4 – 0.5 µg lambda DNA digested with TsoI, probe prepared for loading with 6X DNA Loading Dye & SDS Solution
5 – 0.4 µg of the 2 fragment ligation mixture prior the addition of T4 DNA Ligase
6 – 0.4 µg of the 2 fragment ligation mixture after the ligation with T4 DNA Ligase, probe prepared for loading with 6X DNA Loading Dye
7 – 0.4 µg of the 2 fragment ligation mixture after the ligation with T4 DNA Ligase, probe prepared for loading with 6X DNA Loading Dye & SDS Solution

Non-denaturing Agarose Gel Electrophoresis

Note

Use a flask of at least three times larger 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 or 10X TBE Buffer to a 1X concentration immediately before use.
Use TBE buffer for analysis of DNA bands smaller than 1500 bp. For larger DNA, use TAE buffer.
For intensified gel staining, add ethidium bromide to both the gel and the electrophoresis buffer at a final 0.5 µg/ml concentration. Alternatively, stain the gel after electrophoresis (see below).
Wear gloves when handling ethidium bromide.
For reliable analysis of supercoiled/relaxed plasmid ethidium bromide should not be included in the electrophoresis buffer or gel. The gel should be stained only after electrophoresis is complete.
Ethidium bromide and exposure to UV light may cause DNA alterations. Therefore, avoid UV exposure and do not stain DNA with ethidium bromide if the purified fragments will be used for cloning experiments.

Weigh out the required amount of agarose (depending on the gel percentage) into an Erlenmeyer flask.
Add the appropriate volume of either 1X TBE or 1X TAE buffer and swirl to mix.
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%.
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 3-5 min 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.
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: for intensified gel staining add ethidium bromide to a final concentration of 0.5 µg/ml. Mix well and heat for 1 min without boiling.
Cool the solution to 65-70°C. 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.
Solidify the gel for approximately 30 min before use. Low percentage LM agarose gels need to be solidified at 4°C.
Immerse the gel into the desired electrophoresis buffer. Load the samples onto the gel.
Run electrophoresis at 5-7 V/cm until the bromophenol blue runs approximately two-thirds of the way down the gel.
After electrophoresis the gel can be stained by immersing it into a 0.5 µg/ml ethidium bromide solution for 15-20 min, stained with SYBR® Green I or any other DNA staining technique.
Warning. Hot agarose solution should be handled very carefully.

Alkaline Agarose Gel Electrophoresis

Note

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 larger volume than that of the solution to avoid boiling over.
Wear gloves when handling ethidium bromide.

Weigh out the required amount of agarose (depending on the gel percentage) into an Erlenmeyer flask.
Add the appropriate volume of the buffer (30 mM NaCl, 2 mM EDTA, pH 7.5) and swirl to mix.
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%.
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 3-5 min 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.
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.
Cool the solution to 65-70°C. 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.
Solidify the gel for approximately 30 min before use.
Immerse the gel for at least one hour into the alkaline electrophoresis buffer (30 mM NaOH, 2 mM EDTA). Dilute 5 volumes of the DNA sample or ladder with one volume of 6X alkaline electrophoresis loading buffer (180 mM NaOH, 6 mM EDTA, 18% Ficoll 400, 0.05% bromcresol green).
Heat samples and ladder at 70°C for 5 min, then chill on ice for 3 minutes. Load onto the gel.
Run electrophoresis at 3 V/cm in alkaline electrophoresis buffer (30 mM NaOH, 2 mM EDTA) until the dye runs approximately two-thirds of the way down the gel.
After electrophoresis the gel should be immersed for 30 min in 100-300 ml of 0.5 M Tris-HCl buffer, pH 7.5 and later stained in a 0.5 µg/ml ethidium bromide solution for 30 min. If staining is not enough, the whole procedure can be repeated.

Warning. Hot agarose solution should be handled very carefully.

DNA 5'-end Labeling by T4 Polynucleotide Kinase in the Exchange Reaction

All types of DNA ends can be successfully labeled with T4 Polynucleotide Kinase. However, the labeling efficiency is greatest for the 5'-protruding DNA ends, lower for blunt ends, and is the lowest for 5'-recessed DNA ends.
This protocol is recommended for radiolabeling of DNA markers and ladders. (Ready-to-use versions with the loading dye pre-added are not suitable for labeling).

Prepare the following reaction mixture:

Total volume	20 µl
digested DNA	1-20 pmol of 5'-termini
10X reaction buffer B for T4 Polynucleotide Kinase	2 µl
[gamma-³²P or gamma-³³P]-ATP	40 pmol
24% (w/v) PEG 6000 solution	4 µl
Water, nuclease-free	to 19 µl
T4 Polynucleotide Kinase	1 µl (10 u)

Incubate at 37°C for 30 min.
Add 1 µl 0.5 M EDTA, pH 8.0 and heat at 75°C for 10 min.
Separate labeled DNA from unincorporated label by gel filtration on Sephadex G-50.

Note

If an ethanol solution of [gamma-³²P or gamma-³³P]-ATP is used, dry the required amount of ATP under vacuum and dissolve in Water, nuclease-free (#R0581).
The ATP concentration should be at least 2 µM in the exchange reaction (1, 2).
For estimation of pmol of DNA ends, see REviewer™.

References

Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the Third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M., et al., ed.), John Wiley & Sons, Inc., Brooklyn, New York, 3.10.2-3.10.5, 1994-2004.

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