Nucleic Acid Gel Electrophoresis and Blotting Support—Getting Started
Find valuable information.
Optimize your experiments to get the best results. We’ve compiled a detailed knowledgebase of the top tips and tricks to meet your research needs.
View the relevant questions below:
Agarose is commonly used as it is nontoxic, easy to use, and offers a broad range of separation. We offer precast E-Gel® Agarose Gels or reagents to pour your own agarose gels. Polyacrylamide gels are typically used for high resolution of DNA molecules that range in size from 10–3,000 bp. We offer precast Novex® TBE polyacrylamide gels and UltraPure™ reagents.
For nondenaturing RNA electrophoresis, we recommend using our E-Gel® Precast Agarose Gels. Please note that E-Gel® Agarose Gels are not validated to be RNAse-free. However, many of our customers routinely use E-Gel® Agarose Gels for RNA analysis with success. If RNA is run on an E-Gel® Agarose Gel, any loading buffer that would be used for nondenaturing RNA electrophoresis should be fine.
For denaturing RNA electrophoresis, there are several denaturing agents to choose from, including formaldehyde, glyoxal, formamide, and methyl mercury. Denaturing conditions disrupt hydrogen bonding so that RNA runs without secondary structure, as single-stranded molecules.
For denaturing RNA electrophoresis, our E-Gel® EX Agarose Gels can be used. The only denaturing agent that is compatible with the E-Gel® EX system is formamide, 50-90%. Using other denaturing agents will result in poor band separation and morphology. Please note that we do not recommend running samples prepared in RNA loading buffer on the same gel with samples prepared in water. Please see below for the RNA loading buffer recipe and denaturing electrophoresis conditions:
RNA Loading Buffer:
Deionized formamide: 200 µl
10X MOPS-EDTA-Sodium Acetate Buffer (0.4 M MOPS, pH 7.0, 0.1 M sodium acetate, 10mM EDTA): 40 µl
Deionized formaldehyde: 76 µl
Water: 14 µl
Denaturing Electrophoresis Conditions:
1. Mix 15 µl of RNA loading buffer with 1-5 µl of RNA (1-5 µg).
2. Heat samples at 65 degrees C for 10 min to denature RNA.
3. Place samples on ice immediately after heating.
4. Load entire sample onto an E-Gel® EX agarose gel.
5. Electrophorese for 30 minutes.
For denaturing RNA electrophoresis under formaldehyde-free conditions, we recommend using our NorthernMax®-Gly Kit (Cat. No. AM1946). With this kit, RNA samples are denatured in glyoxal/DMSO loading buffer and run on a glyoxal-containing agarose gel.
Intact RNA should have a 2:1 ratio of 28S:18S bands. You may see a smear of RNA that extends from <9 kb to 0.5 kb, indicating the presence of mRNA in the sample. To see an image or to read more about RNA assessment, click here.
Using glyoxal/DMSO instead of formaldehyde avoids the need to pour and run gels in a fume hood and eliminates safety issues associated with formaldehyde.
The percentage gel to use depends on the size of the molecule you wish to view.
The E-Gel® system for E-Gel® SYBR® and ethidium bromide gels is a precast bufferless TAE system that uses ion exchange matrices. The gels themselves are enclosed by a semi-UV-transparent cassette.
To create a bufferless system, each E-Gel® cassette contains two unique ion exchange matrices that lie between the running gel and the electrodes. The ion exchange matrices provide a buffer-ion reservoir that supplies a continuous flow of acetate, Tris, and ethidium ions throughout the gel. This patented technology results in a sustained electric field with enhanced buffering capacity. E-Gel® gels prevent your direct exposure to ethidium bromide and eliminate the need for you to prepare and dispose of liquid buffer, thus saving time and waste.
Unfortunately, you cannot order the E-Gel® PowerBase™ v.4 as a standalone item. However, this E-Gel® base and power supply is available in some of the E-Gel® starter packs, including the following:
- E-Gel® 1.2% Agarose Gels with SYBR® Safe™ Starter Kit, 6 gels and E-Gel® PowerBase™ v.4, Cat. No. G6206-01
- E-Gel® 2% Agarose Gels with SYBR® Safe™ Starter Kit, 6 gels and E-Gel® PowerBase™ v.4, Cat. No. G6206-02
- E-Gel® 0.8% Agarose Gels with Ethidium Bromide Starter Pak, 6 gels and E-Gel® PowerBase™ v.4, Cat. No. G6000-08
- E-Gel® 1.2% Agarose Gels with Ethidium Bromide Starter Pak, 6 gels and E-Gel® PowerBase™ v.4, Cat. No. G6000-01
- E-Gel® 2% Agarose Gels with Ethidium Bromide Starter Pak, 6 gels and E-Gel® PowerBase™ v.4, Cat. No. G6000-02
We recommend the E-Gel® Opener (Cat. No. G530001) for the SYBR® and ethidium bromide E-Gel® Agarose Gels. The E-Gel® EX Agarose Gels, E-Gel® SizeSelect™ Agarose Gels and E-Gel® GO! Agarose Gels can be opened with the Gel Knife (EI9010). We do not recommend opening the E-Gel® 48 Agarose Gels or the E-Gel® 96 Agarose Gels.
E-Gel® EX Agarose Gels separate DNA faster, offer enhanced sensitivity, and provide added flexibility. The stain within the EX gels is proprietary (not based on SYBR® technology), though it has the same spectral properties as SYBR® stains. The E-Gel® EX gels requires the E-Gel® iBase™ Power System for operation, while E-Gel® Agarose Gels can be run on both the PowerBase™ v.4 or the E-Gel® iBase Power System. The E-Gel® EX gels have a sensitivity that is 5X greater than that using ethidium bromide.
E-Gel® EX Agarose Gels can be used for either DNA or RNA. RNA separation occurs under nondenaturing or denaturing conditions. Please note, our gels are not QC tested for the presence of RNases. See our suggestions below for running your nondenaturing or denaturing samples:
1. Mix RNA sample with 15 µL of RNase-free water.
2. Do not heat. Load the entire sample onto the E-Gel® EX Agarose Gel.
3. Electrophorese for 30 minutes.
1. Mix 15 µL of RNA loading buffer with 1–5 µL of RNA (1–5 µg).
2. Heat samples at 65°C for 10 minutes to denature RNA.
3. Place samples on ice immediately after heating.
4. Load entire sample onto E-Gel® EX Agarose Gel.
5. Electrophorese for 30 minutes.
*The only denaturing agent that is compatible with the E-Gel® EX system is formamide, 50–95%. Heating the sample for 5 minutes at 65°C should be sufficient for denaturing. Using other denaturing agents like glyoxal, formaldehyde, or urea will result in very poor separation and band morphology on E-Gel® EX Agarose Gels. Additionally, we do not recommend running samples with RNA loading buffer on the same gel as samples loaded with water.
We offer our E-Gel® SizeSelect™ Agarose Gels as well as our E-Gel® CloneWell™ Agarose Gels, which are double-comb, precast agarose gels with simplified DNA recovery. Load your sample into the top row and electorphorese until your band or desired size range enters the bottom row. Then, easily remove the size-selected DNA with a pipette. No additional gel purification steps are necessary.
Yes, please ensure that the second row is filled with sterile water prior to running your band of interest into the collection well. Please note that the refill volume may vary between wells. Do not overfill.
No, we have tested E-Gel® Agarose Gels with water in all lanes and with water in just the lanes with sample and observed no apparent differences. But for E-Gel® CloneWell™ Agarose Gels and E-Gel® SizeSelect™ Agarose Gels, it is important to add the water according to the manual.
High-throughput E-Gel® Agarose Gels have staggered wells, and are based on a neutral-pH internal buffer system as opposed to an ion exchange matrix. High-throughput E-Gel® Agarose Gels cannot be opened, and should be run on the E-Gel® E-Base™ system instead of the iBase™ or PowerBase™ systems.
Our UltraPure™ Agarose is standard melting-point agarose designed for routine separation and analysis of DNA and RNA fragments in the 500–23,000 range. UltraPure™ Agarose 1000 is a specialized agarose that provides higher resolution of PCR fragments and other short DNA fragments. We also offer an UltraPure™ Low Melting Point Agarose, which is ideal for resolving DNA fragments from 10 to 1,000 bp with a low melting temperature of 65°C or less.
E-Gel® 48 and E-Gel® 96 gels contain a proprietary neutral-pH internal buffer system with special high capacity and low conductivity features. There are no ion exchange matrices.
The EG program is to run E-Gel® 96 and 48 gels, while the EP is to run the E-PAGE™ 96 and 48 gels.
The mother base plugs into the wall, while daughter bases attach to the mother base. Up to 20 daughter bases can be attached to 1 mother base for a simultaneous run.
We offer our E-Editor™ Software, which can help you align images after a gel run. The E-Editor™ 2.0 Software is only available for PCs, but the older E-Gel® 96 Editor software is still available for the Mac® operating system and can align images from E-Gel® 96 and E-PAGE™ 96 gels. However, the original software is not compatible with E-Gel® 48 or E-PAGE™ 48 gels. Please go to www.thermofisher.com and enter "E-Editor software" in the main search to download the E-Editor™ Software. You can use the E-Gel® Imager System for data analysis.
Upon microwaving, the ReadyPouch™ Agarose solution starts to boil at ~90 seconds and continues as a gentle boil inside the pouch. Even after 5 minutes, the liquid does not boil over. The liquid will eventually evaporate if microwaved for a long time.
The plastic pouch containing the ReadyPouch™ Agarose is a poor heat conductor. Unlike glass, the plastic pouch stays cool upon microwaving, so when the agarose starts to climb the sides of the pouch, it hits a cool spot and the vigorous boil subsides. Secondly, plastic is not as rigid and smooth as glass. Imperfections in the interior surface create a very slightly rough surface area that allow for a gentle boil rather than the explosive, large bubbles obtained with a smooth surface.
The gel strength of a 1% ReadyPouch™ Agarose gel is ≥ 1,200 g/cm2.
ReadyPouch™ Agarose is a standard melting temperature, multi-purpose agarose ideal for routine separation resolving DNA and RNA fragments from 100-8,000 bp.
The shelf life of ReadyPouch™ Agarose is 6 months with a recommended storage temperature of 15 degrees C to 30 degrees C (60-80 degrees F).
TAE buffer is the most commonly used running buffer for medium- and larger-sized PCR amplicons. The borate in TBE is a known enzyme inhibitor and can interfere with downstream enzymatic applications such as ligation, cloning, and protein expression that require DNA purified from agarose gels. Additional concentrations and buffers may be considered for future product line expansion.
Ethidium bromide is supplied in a plastic dropper containing 1 mL at a concentration of 0.625 mg/mL. We recommend using 2 drops/pouch for a final in-gel concentration of 0.5 µg/mL.
It is not advisable to reheat the leftover ReadyPouch™ agarose in the pouch once it has been used. There is a volume loss of ~10% each time the agarose solution is microwaved (assuming that the solution is microwaved for the same amount of time).
E-Gel® Imager – gel documentation
There are three base options for the E-Gel® Imager:
- Blue-light transilluminator base—ideal for innovative DNA stains such as SYBR® Safe™stains, SYBR® Green stains, and those found in E-Gel® EX Agarose Gels.
- UV transilluminator base—best for gels traditionally stained with ethidium bromide.
- E-Gel® adapter base—a one-of-a-kind, real-time gel documentation base that’s compatible with the E-Gel® Go!™ System or E-Gel® iBase™/E-Gel® Safe Imager™ System.
Yes, you will need to purchase the E-Gel® Imager White-Light Conversion Screen (Cat. No. 4473061), that converts blue light emitted by the blue-light transilluminator or UV light emitted by the UV-light transilluminator to white light. This conversion screen is compatible with multiple protein stains including SimplyBlue™ SafeStain, SilverQuest™ silver stain, and Coomassie® blue stains.
Gels stained with SYPRO® Ruby Protein Gel Stain do not need the white-light conversion screen. They can be visualized using a E-Gel® Imager Qdot® 625 Filter (Cat. No. 4466607).
These E-Gel® Imager Filters are alternative filters that can be easily utilized by removing the universal filter tray and sliding in the filter tray of your choosing. We offer an orange E-Gel® Imager Universal Filter, a green filter optimal for SYBR® Green and SYBR® Safe™ stains and those found in E-Gel® EX Agarose Gels, and a red filter for use with our Molecular Probes® Qdot® 625 products.
The E-Gel® Go! Base is a low-throughput, programmable device designed to allow electrophoresis of E-Gel® Go! Agarose Gels, which contain 4 lanes per gel. The gels are offered as 1% or 2% agarose, with a proprietary DNA stain. You can run the gels in 15 minutes for full separation. The system offers the option of a portable battery pack or car adapter for convenience.
Our E-Gel® iBase™ Power System is compatible with all E-Gel® Agarose Gels, including the E-Gel® CloneWell™, E-Gel® SizeSelect™, and E-Gel® EX gels except for the E-Gel® 96 or E-Gel® 48 gels.
Our E-Gel® PowerBase™ v.4 system is compatible with the E-Gel® Agarose Gels with SYBR® Safe™ stain or ethidium bromide (both single and double comb).
The recommended load amount of DNA per band/per well is 20–100 ng. No more than 500 ng DNA per band should be used for most E-Gel® Agarose Gels, with the exception of the E-Gel® with SYBR® Safe stain, which can handle up to 700 ng per lane.
The E-Gel® Safe Imager™ Real-Time Transilluminator docks onto the E-Gel® iBase™ Power System and allows real-time imaging of the migration of DNA/RNA in E-Gel® Agarose Gels containing SYBR® Safe™ stain. The Safe Imager™ 2.0 Blue-Light Transilluminator is an independent system designed for viewing stained gels on the benchtop.
Please see the instrument specifications below:
- Instrument dimensions: 195 × 325 × 65 mm (11.6 × 12.8 × 2.6 in)
- Viewing surface dimensions: 190 × 190 mm (7.5 × 7.5 in)
- Light source: light emitting diodes (LED) producing a narrow emission peak centered at ~470 nm
- LED life: 50,000 hours
- Complies with the European Community Safety requirements
- Contains Class 1 LED products
- Included accessories: amber filter unit, viewing glasses, and international power cord
HR stands for higher resolution. This run lasts 30 minutes and is typically used to separate bands that are similar in size.
Loading buffer is optional. Samples can be loaded directly into the wells if no buffer is used or you can dilute them with deionized water or TE buffer. If you want to use a loading buffer, please see the recipes below:
E-Gel® Agarose Gels (including EX)
10 mM Tris-HCl, pH 7.5
1 mM EDTA
0.005% bromophenol blue
0.005% xylene cyanol FF
E-Gel® CloneWell™ and E-Gel® SizeSelect™ gels
10 mM Tris-HCl, pH 7.5
1 mM EDTA
Alternatively, you can use 10X BlueJuice™ Gel Loading Buffer or TrackIt™ Loading Buffer. Dilute this buffer 50- to 200-fold to obtain optimal results with E-Gel® Agarose Gels.
Here’s a suggested protocol:
- Prewet a nylon membrane suitable for use with RNA using 5X SSC buffer.
- Using the E-Gel® Opene,r remove the E-Gel® Agarose Gel from the cassette.
- Soak gel 2 times for 10 minutes in 5X SSC, 10 mM NaOH at room temperature.
- Using standard techniques, assemble a capillary transfer device using 5X SSC, 10 mM NaOH as the transfer buffer.
- Transfer should be complete after 2 hours. Remove the membrane from the transfer setup.
- Rinse the membrane for 5 minutes in 5X SSC.
- Place the membrane on filter paper to dry (2–4 minutes). Bake the membrane for 30 minutes at 80°C under vacuum or fix RNA to the membrane using a UV crosslinker.
- Place the membrane between two pieces of blotting paper and seal in a hybridization bag. Store in a cool dry place.
Here are the specifications:
- Connectivity: USB2.0
- Camera power supply: AC: 100–240V, 50–60 Hz; DC: 7.5 V 2.0 A
- Camera hood dimensions: 35.6 cm (height) × 28.4 cm (length) × 20.3 cm (width)
- Electrical requirements: 100–240 V, 50/60 Hz, 0.6 A
- Temperature: Ambient ± 5°C to 40°C
- Base dimensions: 11.9 cm (height) × 30.4 cm (length) × 21.4 cm (width) (this is the entire base)
- Viewing surface dimensions: 15 cm x 12 cm (this is the area where the gel is placed)
- Adaptor specifications: Use only the UL Listed adaptor supplied with the E-Gel® Imager Camera Hood (100–240 VAC, 50/60 Hz, 0.6 A)
- Weight: 1 kg
Both types of software come with each E-Gel® Imager system. GelCapture™ software is used to control the camera hood during image acquisition and to perform basic manipulations to the image once acquired. The GelQuant™ Express software is used to analyze images after they have been acquired. This includes estimating the size and relative or absolute mass of bands of interest as well as providing a data report for an entire gel if needed. GelQuant™ Express software can only be used on a given computer while the activation dongle (HASP key) is inserted into a USB port.
For our E-Gel® Imager, we recommend using the UV light base with the E-Gel® Imager Universal Filter for ethidium bromide gels, and either the blue light base with the E-Gel® Imager Universal Filter or the UV light base with E-Gel® Imager UV/SYBR® Filter (green filter) for SYBR® gels.
Robotic scripts for running E-Gel® 96 Agarose Gels and E-PAGE™ 96 Gels on Beckman Coulter's Biomek® FX workstation can be found on our website at: www.thermofisher.com/egels (click the Labware Definitions link in the left navigation pane).
Please see the recipes below.
TBE Running Buffer (5X):
Tris base, 54.0 g
Boric acid, 27.5 g
EDTA (free acid), 2.9 g
Deionized water to 1.0 L
Hi-Density TBE Sample Buffer (5X):
5X TBE Running buffer (Cat. No. LC6675), 2 mL
Ficoll® Type 400, 1.5 g
Bromophenol blue, 1 mL of 1% solution
Xylene cyanol 1 mL of 1% solution
Deionized water to 10.0 mL
The shelf life is 8 weeks for the gels, which should be stored at 4°C.
Please see the gel specifications below:
Gel matrix: Acrylamide/bis-acrylamide
Gel thickness: 1.0 mm and 1.5 mm
Gel size: 8 cm x 8 cm (height x width)
Cassette size: 10 cm x 10 cm (height x width)
Voltage: 200 V constant*
Approximate current at start: 10–18 mA/gel
Approximate current at end: 4–6 mA/gel
Run time: Approximately 30–90 minutes, dependent on gel percentage. The run is complete when the bromophenol blue (darker) tracking dye reaches the bottom of the gel.
* Voltages up to 250 V may be used to reduce run time.
Yes, for ethidium bromide staining, soak the gel in a 2 µg/mL solution of ethidium bromide in ultrapure water for 20 minutes. Destain by rinsing with three successive 10-minute rinses of ultrapure water. Visualize bands under UV light.
The EDTA concentration in our TBE gels is 0.06% (w/v). The 20% TBE gels contain 4% glycerol for maximal resolution. All other TBE gels contain 0.8% glycerol in a layer that represents the bottom 9% of the gel. There is no glycerol in the rest of the gel.
On the Novex® 10% TBE gels, a 51 bp marker can be clearly seen. On the Novex® 20% TBE gel, the 18 and 12 bp markers can be clearly seen.
There are many sample buffer formulations used; however, we have found a distinct difference in the band appearance depending on the sample buffer composition. After evaluating urea, formamide, and various buffer systems, we found that the sharpest, flattest bands were obtained with a urea, Ficoll®, and TBE buffer solution. Sample buffers made with formamide provided fuzzy, indistinct bands.
We provide two optimized TBE-urea sample loading buffers: the Novex® TBE-Urea Sample Buffer contains two tracking dyes, while the Novex® Prep TBE-Urea Sample Buffer contains no tracking dyes. The first is used for analytical runs, and the prep sample buffer without marker dyes is used when UV shadowing will be used prior to cutting out the band or when the marker dyes may interfere with the oligonucleotide bands.
The Novex® 6% DNA Retardation Gels contain 0.5X TBE. Both gels will work for gel retardation; however, the 1X TBE in the Novex® 6% TBE Gels have a higher ionic environment, which may affect DNA-protein interactions. The 0.5X TBE used in the Novex® 6% DNA Retardation Gels usually works better, as it offers good fragment separation in electrophoresis yet has an ionic strength low enough to promote DNA-protein interactions.
A shift assay is a DNA-binding assay using nondenaturing PAGE. It provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-binding proteins. Proteins bind specifically to an end-labeled DNA fragment corresponding to the individual protein-DNA complexes. You can use the assay to test binding of purified proteins or of uncharacterized factors in crude extracts. This assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and binding specificity of DNA-binding proteins.
A supershift assay is a variation of the mobility shift DNA-binding assay that uses antibodies to identify proteins present in the protein-DNA complex.Addition of a specific antibody to a binding reaction can have one of several effects. If the protein recognized by the antibody is not involved in complex formation, addition of the antibody should have no effect. If the protein that forms the complex is recognized by the antibody, the antibody can either block complex formation or it can form an antibody-protein-DNA ternary complex and thereby specifically result in a further reduction in the mobility of the protein-DNA complex (a supershift). Results may be different depending upon whether the antibody is added before or after the protein binds DNA (particularly if there are epitopes on the DNA binding surface of the protein).
These are our suggestions:
- Because alkaline transfer can overhydrolyze small RNAs, and hence decrease their binding, do not exceed 4 hours of transfer.
- Pour gels as thin as possible (usually between 5–6 mm).
- We recommend 15–20 minutes of transfer time per millimeter of gel thickness.
- Crosslinking (using UV) or baking (100°C for 10 minutes) is essential to assure that nucleic acids are irreversibly bound to the membrane.
ULTRAhyb®-Oligo solution contains 25% formamide and is only compatible with positively charged or neutral nylon membranes.
We recommend following the instructions in our mirVana™ miRNA Isolation Kit (Cat. No. AM1560, AM1561) for miRNA Northern blotting. We recommend starting with 2 µg total RNA or a 1 µg miRNA-enriched sample. Run your samples on a denaturing polyacrylamide gel, as the RNA may be too small to detect on an agarose gel. Transfer RNA to a nylon membraneby electroblotting. The compositions of recommended prehybridization, hybridization, and wash buffers are provided in the manual.
Yes, we offer our mirVana™ miRNA Probe Construction Kit (Cat. No. AM1550) and our mirVana™ Probe and Marker Kit (Cat. No. AM1554).
Yes. You can use a 15% denaturing polyacrylamide gel for Northern analysis of small RNAs, such as miRNAs and siRNAs. A hyridization buffer optimized for use with short probes, such as ULTRAhyb®-Oligo solution, should be used for the best results. For more sensitive detection, enrich the RNA sample for small RNAs (e.g., with the mirVana™ miRNA Isolation Kit or mirVana™ PARIS™ RNA and Native Protein Purification Kit. Of course, using a solution hybridization assay such as the mirVana™ miRNA Detection Kit to analyze small RNAs can provide much greater sensitivity and allow you to detect multiple small RNAs simultaneously.
Running small 10 cm gels for Northern blotting takes 30–90 minutes, much quicker than larger gels. The biggest time savings, however, can be during transfer to the membrane. Traditionally, Northerns have been blotted overnight using capillary transfer and a high-salt buffer (10X SSC or 10X SSPE). By using a weak base as the medium (e.g., NorthernMax® One-Hour Transfer Buffer), the transfer can be completed in just 1 hour. Alternatively, you can electroblot your RNA in 1 hour.
For Research Use Only. Not for use in diagnostic procedures.