Pre-Cast Gels for Low-Throughput Nucleic Acid Analysis

The E-Gel agarose gel electrophoresis system is a complete bufferless system for agarose gel electrophoresis of DNA samples.

The major components of the system are:

• E-Gel pre-cast agarose gels
• E-Gel PowerBase v.4. or Mother E-Base

E-Gel pre-cast agarose gels are self-contained gels that include electrodes packaged inside a dry, disposable, UV-transparent cassette. The E-Gel agarose gels run in a specially designed device that is a base and power supply combined into one device. They either contain SYBR Safe™ DNA gel stain, ethidium bromide, or no DNA gel stain.
 
Advantages of E-Gel
Using E-Gel agarose gels for electrophoresis of DNA samples offer the following advantages:

• Provides fast, safe, consistent, high-resolution electrophoresis
• Eliminates the need to prepare agarose gels and buffers, and to stain gels
• Compatible with most commercially available robotic systems for high-throughput agarose gel electrophoresis
• Available in a variety of agarose percentages, well formats, and throughput to suit your applications
• Offered with SYBR Safe™ DNA gel stain, a safer, more environmentally friendly alternative to ethidium bromide

Available DNA Gel Stains
E-Gel® agarose gels come in three formats for staining your DNA:

• Regular E-Gel agarose gels contain the standard DNA gel stain ethidium bromide
• E-Gel with SYBR Safe™ contains SYBR Safe™ DNA gel stain, which is not classified as hazardous waste under U.S. Federal regulations, and improves cloning efficiency when using blue light for imaging
• Clear E-Gel does not contain a DNA gel stain, and is intended for custom staining or for analyzing fragments coupled to a fluorescent label.

Advantages of SYBR Safe™
SYBR Safe™ DNA gel stain offers the following advantages:

• SYBR Safe™ DNA gel stain is not classified as hazardous waste under U.S. Federal regulations and meets the requirements of the Clean Water Act and the National Pollutant Discharge Elimination System regulations.
• SYBR Safe™ DNA gel stain does not cause mutations, chromosomal aberrations, or transformations in appropriate mammalian test systems, in contrast to ethidium bromide which is a strong mutagen.
• A single oral administration of SYBR Safe™ DNA gel stain produces no signs of mortality or toxicity at a limit dose of 5000 mg/kg.
• Visualizing E-Gel with SYBR Safe™ using blue light transilluminators dramatically reduces DNA damage that lowers cloning efficiency.
• For details on SYBR Safe™ DNA gel stain.

Throughput Formats
Two types of E-Gel agarose electrophoresis systems are available from Invitrogen based on your throughput requirements.

• Low-Throughput E-Gel Electrophoresis System
• Medium/High-Throughput E-Gel Electrophoresis System

Low-Throughput E-Gel Electrophoresis System
The Low-Throughput E-Gel Electrophoresis System is designed for electrophoresis of 12-16 DNA samples per gel. For details on this system, see page 5.The system consists of the following components:

• E-Gel with SYBR Safe™, E-Gel single comb, double comb, and Clear E-Gel pre-cast agarose gels
• Gels are available in a variety of well formats and percentages. Choose an appropriate gel based on your application. (see table).
• E-Gel PowerBase v.4
• The E-Gel PowerBase v.4 is a base and a power supply in one device. The E-Gel PowerBase connects directly to an electrical outlet using the adapter supplied with the base.
• The E-Gel Opener is an easy-to-use device specifically designed to open any E-Gel single comb, double comb, or Clear E-Gels

System Components
The Low-Throughput E-Gel Electrophoresis System consists of the following components:

• E-Gel with SYBR Safe™, E-Gel single comb, double comb, and Clear E-Gel pre-cast agarose gels
• E-Gel PowerBase v.4
• E-Gel Opener

Note:   The E-Gel Base previously available from Invitrogen can be used for electrophoresis of E-Gel with SYBR Safe™, E-Gel single comb, double comb, and Clear E-Gel agarose gels.

Applications
E-Gel agarose gels are suitable for analyzing:

• PCR products
• Restriction digests
• RT-PCR reactions

 
When to Use E-Gel With SYBR Safe™
E-Gel with SYBR Safe™ contains SYBR Safe™ DNA gel stain instead of ethidium bromide. Use E-Gel with SYBR Safe™ for these reasons:

• If you want to minimize your hazardous waste, since SYBR Safe™ DNA gel stain is not classified as such under U.S. Federal regulations.
• If you want to protect yourself or your co-workers, because E-Gel with SYBR Safe™ eliminates the use of the strong mutagen ethidium bromide and reduces UV exposure.
• If you want to maximize cloning efficiency, since E-Gel with SYBR Safe™ dramatically reduces DNA damage if using blue light transilluminators.

When to Use Clear E-Gel
Clear E-Gel agarose gels do not contain ethidium bromide in the gels and are ideal for the following applications:

• Post-staining gels with sensitive stains such as SYBR Green I or II, or SYBR Gold.
• Analyze DNA fragments pre-labeled with fluorescein, Texas Red or Alexa Fluor
• Perform downstream applications with DNA in which ethidium bromide may interfere
• Determine accurate size and form of the DNA band (most DNA staining dyes intercalate into the DNA and change molecular weight and form of the DNA)
• Stain only one lane of the gel and then excise a specific band or size range in the remaining gel for cloning purposes thereby avoiding UV exposure to the DNA resulting in a significant increase in the cloning efficiency.

E-Gel Single Comb and Double Comb Gels
The E-Gel single comb and double comb gels are bufferless gels containing electrodes embedded in the agarose matrix. Each gel contains an ion generating system (TAE buffer system), a pH balancing system, and ethidium bromide for DNA staining and is packaged inside an UV-transparent cassette.
 
To create a patented bufferless system, each E-Gel single comb and double comb cassette contains two ion exchange matrices (IEMs) that are in contact with the gel and electrodes. The IEMs supply a continuous flow of ions through out the gel resulting in a sustained electric field required for running the gel (see figure below).
 
Clear E-Gel agarose gels are single comb gels that do not contain any nucleic acid stain in the agarose.

E-Gel PowerBase
The E-Gel PowerBase Version 4 is an easy-to-use, automated device specifically designed to simplify electrophoresis of single comb or double comb E-Gel agarose gels from Thermo Fisher Scientific. The E-Gel PowerBase is a base and a power supply all in one device.
 
The operation of the E-Gel PowerBase v. 4 is controlled by two buttons on top of the base. The left button is for a double comb run and right button is for a single comb run (see the label on the unit). To select different electrophoresis runs for the PowerBase, do one of the following.

• Press and release the button OR
• Press and hold the button

E-Gel Base
The E-Gel Base previously available from Invitrogen connects to a power supply and is used for electrophoresis of E-Gel single comb, double comb, and Clear E-Gel agarose gels.

E-Gel Single Comb and Double Comb Gel Specifications
The E-Gel cassette is 8 cm x 10 cm and 0.6 cm thick. The thickness of the E-Gel gel is 3 mm and the volume of the gel is 20 ml.

• Single comb gel—Each well is 4.1 mm wide and the space between wells is 1 mm. The running distance is 5.8 cm. Each gel contains 12 lanes.
• Double comb gel—The sample well is 4.6 mm wide and the marker well is 2.8 mm wide. The running distance from each comb is 2.9 cm. Each gel contains two rows of 8 sample wells and 2 marker wells (M). The wells of the double comb gel are compatible for loading with a multichannel pipettor.

E-Gel 48 Gel Specifications
Each E-Gel 48 gel contains 48 sample wells and 4 marker wells (M). 

E-Gel >96 Gel Specifications
Each E-Gel 96 gel contains 96 sample wells and 8 marker wells (M).

The wells of the E-Gel 96 cassette are compatible with a multichannel pipettor or 8, 12, or 96-tip robotic loading devices.

E-Base Specifications
The specifications for Mother E-Base and Daughter E-Base are listed below.

The SBS (Society for Biomolecules Screening) standard 96-well plate format of the E-Base fits on most robotic platforms allowing the loading and °electrophoresis of gels on the E-Base™ directly on the robot.

 
E-Gel PowerBase v.4 Specifications
The specifications for E-Gel PowerBase V.4 are listed below.

 
E-Gel PowerBase Adapter Specifications
The E-Gel PowerBase v.4 is designed for use with an adapter included with the PowerBase. Use only UL Listed Class 2 Direct Plug-in Adapter included with the PowerBase. Input and Output supplied by the adapter are shown in the table below.

E-Gel Base Specifications
The specifications for E-Gel Base are listed below:

• Dimensions:                           12.5 x 13 x 3.5 cm
• Weight:                                     3.18 oz. (90 g)
• Temperature:                          Ambient 15C to 40°C

Introduction
For optimal results, follow the guidelines for preparing your DNA sample as described in this section.
 
Materials Needed

• DNA sample
• Loading buffer (optional)
• Molecular weight markers

Amount of DNA
Use 20-100 ng DNA per band for samples containing one unique band, or up to 500 ng per lane for samples containing multiple bands. If you are unsure how much to use, test a range of concentrations to determine the optimal concentration for your particular sample. Excess DNA will cause poor resolution.
 
Loading Method
The DNA samples are loaded in E-Gel agarose gels using the One-Step Loading or Two-Step Loading method.
 
The One-Step Loading method is routinely used to load E-Gel agarose gels.
The Two-Step Loading method is used to load E-Gel agarose gels, if the One-Step Loading method produces fuzzy or indistinct bands, or the gel was removed from its plastic pouch for more than 30 minutes

Prepare loading buffer and sample volumes based on the loading method as described below.

Total Sample Volume
The recommended total sample volume for each gel type is listed in the table below.
 
Note:   For best results, keep all sample volumes uniform. If you do not have enough samples to load all wells of the gel, load an equal volume of deionized water (E-Gel) or buffer containing the same salt concentration as samples (E-Gel 48/96 gels) into any empty wells.

Loading Buffer
Loading buffer is optional for One-Step Loading method but is required for the Two-Step Loading method for E-Gels.
 
You can load your samples directly into the wells, if no loading buffer is used. If you are using loading buffer, mix the required amount of DNA with the loading buffer.
 
We recommend using a loading buffer with the following formulation:

• For One-Step Loading method, use a loading buffer that yields a final concentration of 10 mM Tris-HCl, pH 7.5; 1 mM EDTA; 0.005% bromophenol blue; 0.005% xylene cyanol FF.
• For Two-Step Loading method, use a loading buffer that yields a final concentration of 10% glycerol (or 6% Ficoll 400) 10 mM Tris-HCl, pH 7.5; 1 mM EDTA; 0.005% bromophenol blue; 0.005% xylene cyanol FF

If you are using 10X BlueJuice Gel Loading Buffer or TrackIt Loading Buffer available from Invitrogen, dilute this buffer 50-to 200-fold to obtain optimal results with E-Gel agarose gels. For Two-Step loading with 10X BlueJuice Gel Loading Buffer or TrackIt Loading Buffer dilute this buffer 50- to 200-fold and add 10% glycerol.
 
High Salt Samples
Important: To obtain the best results, prepare your high salt samples as described below.
 
Samples containing > 50 mM NaCl, 100 mM KCl, 10 mM acetate ions, or 10 mM EDTA (i.e. certain restriction enzyme and PCR buffers) will cause loss of resolution on E-Gel agarose gels. Dilute samples 2- to 20-fold as described:

• Restriction Digests—Digest 500 ng-1 µg of DNA in 20 µl final volume. Use 1 µl (for samples >1 kb) or 5-10 µl (for samples <1 kb) and dilute as described below.
• PCR Samples—Use 1-5 µl of a 50 µl reaction and dilute as described below.

Preparing Samples
Prepare your samples based on the loading method used as described below: 

DNA Molecular Weight Markers
We recommend using the following DNA molecular weight markers for different types of E-Gel agarose gels to obtain good resolution.
 
Note:   Supercoiled DNA molecular weight markers may produce a slightly fuzzy pattern when run on E-Gel agarose gels containing ethidium bromide. Consider using Clear E-Gel agarose gels and post-staining with ethidium bromide.

Introduction 
After you have prepared your samples, you are ready to proceed with electrophoresis. Instructions are provided below to load and run E-Gel single comb, double comb gels, and Clear E-Gel gels using the E-Gel PowerBase v.4.
 
For details on using the E-Gel agarose gels with the E-Gel Base. The total run times are:

• 30–33 minutes for single-comb/Clear E-Gel gels
• 15–17 minutes for double-comb gels

Pre-running E-Gel with SYBR Safe™
You must first pre-run the E-Gel with SYBR Safe™ agarose gel for 2 minutes with the comb in place before loading your samples to ensure proper resolution of your DNA fragments.
 
Each E-Gel cassette is supplied individually wrapped and ready for use. To set up and use an E-Gel with SYBR Safe™ gel, follow the instructions below:

1. Plug the E-Gel PowerBase v.4 into an electrical outlet using the adapter plug.
2. Open the package containing the gel and insert the gel (with the comb in place) into the apparatus right edge first. Press firmly at the top and bottom to seat the gel in the base. You should hear a snap when it is in place. The Invitrogen logo should be located at the bottom of the base, close to the positive pole. See the diagram below. A steady, red light will illuminate when the E-Gel gel is correctly inserted (Ready Mode).
3. Press and hold either button until the red light turns to a flashing green light. This indicates that the 2-minute pre-run has started.
4. At the end of the pre-run, current will automatically shut off. The flashing green light will change to a flashing red light and the PowerBase™ will beep rapidly.
5. Press and release either button to stop the beeping (you will hear only one beep). The light will change from a flashing red light to a steady red light.

 
Method of Loading Samples
The E-Gel agarose gels are designed for loading samples manually or using a multichannel pipettor. We recommend the following methods of sample loading based on the gel type:
 
Gel Type                                                Method of Loading
 
E-Gel single comb/Clear E-Gel         Manual
E-Gel double comb                             Manual or multichannel pipettor
 
Loading E-Gel
All wells in the gel must contain sample or water. Avoid introducing bubbles while loading, as bubbles will cause bands to distort.

1. Remove the comb from the E-Gel gel using both hands to lift the comb gently by rolling the comb slowly towards you. Be careful to pull the comb straight up from both sides. Do not bend the comb. Remove any excess fluid using a pipette.
2. Load samples into the wells using the One-Step Loading or Two-Step Loading method.
   • One-Step Loading Method: Load 20 µl of sample into each well(page 38 for sample preparation). Load 20 µl of water into any remaining empty wells.
   • Two-Step Loading Method: First load 10 µl of deionized water into each well (including sample and empty wells). Do not premix with sample. Then load 10 µl of sample containing 10% glycerol into each sample well.

Load 100-250 ng of the appropriate molecular weight markers.

Electrophoresis of E-Gels

1. Choose between a 30-minute run for single-comb gels and a 15-minute run for double-comb gels on the E-Gel PowerBase v.4. For the 30-minute run, press and release the 30-min button to start the 30-minute electrophoresis run. The light will change to a steady green light.
   For the 15-minute run, press and release the 15-min button. A steady blue light appears to indicate the beginning of the 15-minute run.
   Note:   The actual running time of the E-Gel gel may vary between 15-17 minutes for double-comb gels and 30-33 minutes for single-comb gels.
2. Current through the E-Gel gel automatically shuts off at the end of each run. The E-Gel PowerBase v.4 signals the end of the run with a flashing red light and rapid beeping.
3. Press and release either button to stop the beeping. The light will turn to a steady red light.
4. At the end of the run, remove the gel cassette from the power unit and analyze your results using a UV transilluminator. For Clear E-Gels, open the gel cassette as described below and proceed to staining the gel (see below).

E-Gel agarose gels can only be used once. Do not re-use them.
 
Staining Clear E-Gel
Clear E-Gel gels are suitable for post-staining DNA bands using a wide variety of DNA stains including SYBR Green I or II, or SYBR Gold.
 
To open the Clear E-Gel cassette for staining, see next page. General guidelines are given below for post-staining DNA on Clear E-Gel gels using a stain of choice. For details, follow the manufacturer’s recommendations.

• For optimal staining sensitivity, make sure the pH of the staining solution is between pH 7.0-8.0.
• Use TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8) buffer to dilute the stain. 

Introduction
The E-Gel Opener is an easy-to-use device specifically designed to open any E-Gel single comb or double comb gels for excision of DNA fragments or for blotting.
 
Do not use the E-Gel Opener to open the E-Gel 48 or 96 cassettes. The E-Gel 48 or 96 cassette cannot be opened.
 
Description of Opener
The E-Gel Opener consists of an anodized aluminum platform housing two recessed steel blades, one which is stationary and one which is movable. The blades are brought into contact with the E-Gel cassette by turning the large knob clockwise.

• Before using the E-Gel Opener for the first time, we recommend that you practice opening a few used E-Gels to familiarize yourself with the process. Practice on E-Gels that will not be used further for preparative purposes.
• Electrophoresis must be complete before opening the E-Gel. We recommend that you place the E-Gel on the transilluminator and photograph the gel before proceeding further. If you plan to isolate DNA from the E-Gel, we recommend that you open the gel and excise the gel fragment immediately after electrophoresis as bands will diffuse within 20 minutes. If you plan to blot the gel, keep your blotting apparatus ready before opening the gel.

The blades on the E-Gel Opener are extremely sharp. Do not insert your fingers into the area housing the blades! Pick up the E-Gel Opener by holding the large knob only (see Figure 1 above). Exercise caution when handling and cleaning the E-Gel Opener. Dispose of blades in a needle disposal container or a Sharps disposal box.
 
Procedure
The following section provides instructions to open an E-Gel cassette. Before beginning, you should wear safety goggles and gloves.

1. Place the E-Gel Opener on a flat surface, with the knob extending off the edge of the laboratory bench and facing the user. Set the E-Gel Opener to its widest open position by turning the knob counterclockwise.
2. Insert the E-Gel into the E-Gel Opener so that two opposing sides of the gel cassette are aligned with the blades. Position the E-Gel such that the two sides fit into the grooves housing the blades.
3. Turn the knob steadily clockwise to bring the blades in contact with the E-Gel cassette. As the knob is tightened, you will hear a series of pops. Continue to turn the knob until the resistance increases. Stop turning the knob as soon as the E-Gel® cassette begins to lift off the surface of the platform. Two sides of the E-Gel will now be unsealed. Note: Once you observe the E-Gel cassette begins to lift off the surface of the platform, do not continue to tighten the knob as you will damage the E-Gel.
4. Unscrew the knob and remove the E-Gel. The E-Gel cassette fits snugly in the recessed groove, and you may have to carefully work the cassette from the housing. Turn the E-Gel 90° and re-insert the gel cassette into the Opener so that the two remaining sealed sides can be opened.
5. Repeat Step 2 to open the remaining two sides of the E-Gel. Stop turning the knob when you see the top of the E-Gel cassette begins to lift off the gel.
6. Unscrew the knob and carefully remove the E-Gel cassette. The 4 sides of the cassette should be unsealed. If not, repeat Steps 2-5 as necessary. Remove the E-Gel and set the opened cassette on your bench.
7. If you plan to blot the gel, do not pick up the gel from the cassette. Lift off the top of the gel cassette. Place the blotting membrane on the gel and pick up the cassette with the gel and membrane. Flip the gel and membrane out of the cassette onto your gloved hand and then flip the gel and the membrane directly onto your wet blotting paper. If you plan to purify DNA from the gel, lift off the top of the gel cassette and excise the gel fragment. Transfer the gel slice to a microcentrifuge tube.
8. After using the opened E-Gel, discard it as hazardous waste.

 
Cleaning and Storage
After use, clean the E-Gel Opener with mild detergent and water to remove excess agarose, ethidium bromide, and plastic from the platform. Use a squirt bottle and wipe the platform dry with a clean tissue. Do not insert your fingers into the area housing the blades, and do not immerse the E-Gel Opener in water as the blades may rust. Store the E-Gel Opener at room temperature.