DNA Ladders & Markers: Introduction, Range Selection and Reference Guides
- Introduction: DNA Electrophoresis (see below)
- Introduction: DNA Ladders & Markers
- DNA Ladders & Markers
Introduction: DNA Electrophoresis
Agarose and polyacrylamide gel electrophoresis is a rapid technique used to identify, quantify and purify nucleic acids. This method, and its variations, are used in a variety of protocols including conventional DNA restriction, PCR analysis, RFLP analysis, genotyping, DNA sequencing, DNA fingerprinting, analyzing DNA-protein interactions, DNA purification, etc.
DNA molecules are negatively charged due to dissociation of the phosphate backbone. During electrophoresis they migrate towards the positively charged electrode. Small DNA fragments migrate more rapidly in the gel matrix compared to large ones. As a result, DNA molecules are separated based on their size.Agarose Gels
Agarose is a non-toxic polysaccharide extracted from seaweed. It is easy to use and is relatively inexpensive if compared to polyacrylamide. A wide range of DNA fragments (10-50,000 bp) can be separated on agarose gels of various concentrations (5-0.4%), but the overall resolving power of agarose gels is relatively low if compared to polyacrylamide.
- Standard high-melting-point agarose (e.g., TopVision™ LE GQ Agarose) is used in routine DNA electrophoresis for separation of a wide range of DNA fragments. Separated DNA fragments can be further extracted from gels using a variety of methods. Fermentas offers the DNA Gel Extraction Kit for silica powder-based DNA purification from agarose gels.
- A low melting/gelling temperature agarose (e.g., TopVision™ LM GQ Agarose) is recommended for rapid DNA extraction from a gel with the agarose digesting enzyme, Agarase. Fermentas DNA Extraction Kit is also suitable for silica powder-based DNA purification from these gels. Low melting temperature agarose is commonly used for “in-gel” DNA treatment with enzymes or for bacterial transformation with nucleic acids directly after re-melting the gel. Low melting point agarose has lower resolving power than that of standard agarose.
Polyacrylamide Gels
Polyacrylamide is a cross-linked polymer of acrylamide. Polyacrylamide gels have a rather small range of separation, but they provide very high resolution of DNA molecules. They are used for the separation of nucleic acids that are less than 500 bp long. Under appropriate conditions, molecules differing in size of a single base pair can be resolved. The typical concentration range for polyacrylamide gels is between 3.5-20%.
Separation of Double-stranded and Single-stranded DNA
Both agarose and polyacrylamide gels can be used for separation of double-stranded and single-stranded DNA molecules.
- Double-stranded DNA molecules can be separated on agarose or polyacrylamide gels under non-denaturing conditions. The size and approximate quantity of the DNA molecules is determined by comparing the DNA band of interest with bands of Fermentas DNA ladders or markers on the same gel. The DNA quantity is well defined for each DNA band in most Fermentas DNA standards.
- Single-stranded DNA molecules are separated on polyacrylamide gels in the presence of strong denaturants (7-8 M urea or formamide) and at high temperature (55°C). Under denaturing conditions, most of the secondary structures of the DNA are eliminated. DNA electrophoresis in agarose gels at alkaline pH is also used to separate single-stranded DNA molecules.
DNA Loading Dye Solutions
Prior to loading the DNA sample on a gel, it is necessary to mix it with an appropriate loading buffer or loading dye solution.
DNA loading dye solutions usually contain:
- Glycerol to ensure that the sample easily sinks into well.
- EDTA, which binds divalent metal ions that may interfere with electrophoresis. By complexing metal ions, EDTA also stops metal-dependent enzymatic reactions, such as DNA degradation by nucleases.
- Electrophoresis tracking dyes (bromophenol blue, xylene cyanol FF, orange G, etc.) to monitor the progress of electrophoresis by the migration of dyes. The concentration of the tracking dyes in Fermentas loading dye solutions is optimized to avoid masking of DNA bands during gel analysis under UV light.
Fermentas offers conventional DNA ladders/markers (dissolved in storage (TE) buffer), as well as ready-to-use versions of the DNA ladders/markers. All ready-to-use versions are premixed with an appropriate loading dye and can be applied directly on the gel. Fermentas loading dyes are supplied with each DNA ladder/marker and are available separately.
Fermentas supplies the following loading dyes:
- 6X DNA Loading Dye with bromophenol blue and xylene cyanol FF for routine electrophoresis.
- 6X MassRuler™ DNA Loading Dye with bromophenol blue for DNA quantification on the gel.
- 6X Orange DNA Loading Dye with xylene cyanol FF and orange G that migrates with the smallest DNA fragments.
- 6X DNA Loading Dye & SDS Solution with bromophenol blue, xylene cyanol FF and 1% SDS for elimination of DNA-protein interactions that may affect DNA migration during electrophoresis.
- 6X TriTrack™ DNA Loading Dye with bromophenol blue, xylene cyanol FF and orange G for a triple tracking of electrophoresis.
Electrophoresis Conditions
The electrophoretic mobility of DNA molecules depends on the voltage and the electrophoresis buffer. The following conditions are recommended for DNA electrophoresis:
- 5-10 V/cm voltage for smaller than 1 kb DNA molecules in both TAE and TBE buffers.
- 4-10 V/cm for 1-12 kb DNA molecules in both TAE and TBE buffers.
- 1-3 V/cm for larger than 12 kb molecules (only in TAE buffer).
Fermentas offers concentrated solutions of Tris-borate and Tris-acetate electrophoresis buffers: 10X TBE Buffer and 50X TAE Buffer.
- TAE Buffer is recommended for resolution of larger than 1.5 kb DNA of supercoiled DNA and for genomic DNA analysis.
- TBE Buffer is recommended for smaller than 1500 bp DNA fragments. It is frequently used for DNA analysis on both native and denaturing polyacrylamide gels.
Gel Staining
Intercalating dyes such as ethidium bromide or SYBR® Green I allow for DNA visualization directly on a gel under UV light. Ethidium bromide can be added to the gel and to the electrophoresis buffer at a final concentration of 0.5 µg/ml. DNA bands look bright under UV in such a pre-stained gel. However, the presence of ethidium bromide in the gel reduces the DNA mobility up to 15%. Therefore, gel staining after electrophoresis is recommended to achieve better resolution of DNA fragments.
By staining with ethidium bromide, up to 10ng DNA can be detected on a gel. The detection threshold on gels stained with SYBR® Green I is about 60 pg of DNA. For detection of femtograms quantity of DNA, Fermentas offers a number of DNA labeling and detection techniques.
Introduction: DNA Ladders & MarkersFermentas offers a broad selection of DNA ladders/markers ranging from 10 bp to 48.5 kb, for accurate analysis of linear double-stranded DNA in agarose or polyacrylamide gels. Most Fermentas DNA ladders/markers are available both in a convenient ready-to-use format (premixed with loading dye solution) and in the conventional format (dissolved in storage (TE) buffer). Our DNA ladders/markers are manufactured and quality controlled to supersede all industry standards, making Fermentas the quality leader for DNA standards.
DNA ladders are made of chromatography-purified individual DNA fragments using a proprietary patent-pending technology for preparation of pharmaceutical grade plasmid DNA.
Fermentas PureExtreme® restriction enzymes are used for the production of DNA fragments for our ladders/markers. As a result, these products are stable during prolonged incubations at room temperature and multiple freeze-thaw cycles.
In addition to DNA ladders/markers Fermentas is offering a new unique opportunity to create the highest quality DNA ladder of desired range and composition from the Collection of Individual DNA Fragments covering the 10-20,000 bp range.
Fermentas offers the NoLimits™ Bulk Custom DNA Ladder Service providing the opportunity to create a ladder of needed range and composition by ensuring the highest quality and performance.Fermentas presents the following groups of DNA ladders/markers:
- NoLimits™ Custom DNA Ladders produced upon request DNA ladders of any combination for a variety of applications.
- Express DNA Ladders, specifically designed for good resolution in short electrophoresis time under various conditions. The quality of the band pattern is not affected by electrophoresis conditions such as composition of the electrophoresis buffer, the voltage or the gel percentage. These new ladders supplement such ladder groups as MassRulers™, GeneRulers™ and compose a new group of ZipRuler™ Express DNA Ladders.
- ZipRuler™ DNA Ladders designed for fast and accurate sizing of broad range DNA fragments.
- FastRuler™ DNA Ladders designed for fast high throughput electrophoresis.
- MassRuler™ DNA Ladders designed for accurate DNA quantification on gels.
- O’RangeRuler™ DNA Ladders are step ladders for precise sizing of DNA fragments differing in 5, 10, 20, 50, 100, 200, 500 bp increments.
- GeneRuler™ & O’GeneRuler™ DNA Ladders designed for sizing and quantification of broad range DNA fragments.
- Conventional Lambda DNA Markers designed for determination of the size of large DNA fragments.
- Conventional Phage & Plasmid DNA Markers are classical markers for small DNA fragments analysis.
- DNA Markers for Genomic DNA Analysis, plasmid DNA-free markers designed for labeling and genomic DNA analysis in Southern blots.
Distinguishing features of Fermentas DNA ladders/markers are the following:
- Allow sizing and quantification of DNA.
- Cover a wide range of DNA fragment lengths.
- Easy-to-remember band sizes and bright reference bands.
- Bright and sharp bands.
- Large packages are provided in small aliquots to minimize the possibility of contamination during usage.
- Ready-to-use DNA ladders/markers can be stored either at room temperature or at +4°C.
- Supplied with DNA loading dye for sample DNA.
- Can be visualized under UV light after ethidium bromide or SYBR® Green I staining.
- Available in bulk quantities.
Main features of Fermentas DNA ladders and markers and their applications are listed in description of each DNA ladder/marker group.
Typical data regarding the purity of the Fermentas DNA ladders and markers is presented in picture below (analyzed with an Agilent 2100 bioanalyzer).
Analysis of MassRuler™ Express LR Forward DNA Ladders and LR Reverse using an Agilent 2100 bioanalyzer and DNA 1000 LabChip® Kit and agarose gel electrophoresis (1% TopVision™ LE GQ Agarose, 10 µl/lane, 8 cm length gel, 1X TAE, 7 V/cm, 30 min).
A - analysis of MassRuler™ Express LR Forward DNA Ladder
B - analysis of MassRuler™ Express LR Reverse DNA Ladder
C - agarose gel analysis of MassRuler™ Express DNA Ladders, LR Forward and Reverse
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Updated vasario 01, 2008 10:36
