Pre-Cast Gels for Safe Nucleic Acid Analysis

SYBR Safe™ DNA Gel Stain
 
SYBR Safe™ DNA gel stain has been specifically developed for reduced mutagenicity, making it safer than ethidium bromide for staining DNA in agarose gels. The detection sensitivity of E-Gel^® with SYBR Safe™ stain is similar to that of E-Gel^® containing ethidium bromide. DNA bands stained with SYBR Safe™ DNA gel stain can be detected using a standard UV transilluminator, a visible-light transilluminator or a laser-based scanner.
 
Safety Features of SYBR Safe™

• SYBR Safe™ DNA gel stain is not classified as hazardous waste under U.S. Federal regulations
• SYBR Safe™ stain meets the requirements of the Clean Water Act and the National Pollutant Discharge Elimination System regulations (though E-Gel^® with SYBR Safe™ generally does not generate liquid waste).
• SYBR Safe™ DNA gel stain does not induce transformations in primary cultures of Syrian hamster embryo (SHE) cells. In contrast, ethidium bromide tests positive in the SHE cell assay, consistent with its known activity as a strong mutagen.
• SYBR Safe™ stain does not cause mutations in mouse lymphoma cells at the TK locus, nor does it induce chromosomal aberrations in cultured human peripheral blood lymphocytes, with or without S9 metabolic activation.
• Compared to ethidium bromide, SYBR Safe™ DNA gel stain causes fewer mutations in the standard Ames test. Weakly positive results occurred in only four out of seven Salmonella strains and only with activation by a mammalian S9 fraction.
• 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.
• View studies documenting the safety of SYBR Safe™ in the SYBR Safe™ White Paper document.

Disposal of SYBR Safe™
 
SYBR Safe™ DNA gel stain is not classified as hazardous waste, but as disposal regulations vary, please contact your safety office or local municipality for appropriate SYBR Safe disposal in your community.
 

Spectrum of SYBR Safe™ Bound to nucleic acids, SYBR Safe™ stain has fluorescence excitation maxima at 280 and 502 nm, and an emission maximum at 530 nm (see figure below).




Normalized fluorescence excitation and emission spectra of SYBR Safe™ DNA gel stain, determined in the presence of DNA

Visualization of SYBR Safe™

Detect DNA bands stained with SYBR Safe™ DNA gel stain using a blue-light transilluminator, a standard UV transilluminator, or a laser-based scanner. For photographing gels, a special filter may be required.

Cloning Benefits of SYBR Safe™
 
Using the SYBR Safe™-blue-light method dramatically reduces DNA damage. As a result, cloning efficiency can improve ten- to thousand-fold.

Introduction
 
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.

 
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. 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.
• 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 adaptor supplied with the base. (page 6). •E-Gel^® Opener
• 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^®.

The Medium/High-Throughput E-Gel^® Electrophoresis System

The Medium/High-Throughput E-Gel^® Electrophoresis System is designed for electrophoresis of 48-96 DNA samples per gel. This system is compatible for use with multichannel pipettors or automated liquid handling systems.

The system consists of the following components:

E-Gel^® 48 gels 

• Each E-Gel^® 48 gel contains 48 sample lanes and 4 marker lanes and is designed for medium-throughput agarose electrophoresis of nucleic acids.

E-Gel^® 96 gels

• Each E-Gel^® 96 gel contains 96 sample lanes and 8 marker lanes in a patented, staggered-well format and is designed for high-throughput agarose electrophoresis of nucleic acids.

E-Base™ Electrophoresis Device

• The E-Base™ is a base and a power supply all in one device and is an easy-to-use, pre-programmed device specifically designed for electrophoresis of E-Gel^® 48 and 96 gels.

E-Holder™ Platform

• The E-Holder™ Platform is designed to hold E-Gel^® 96 gels during robotic loading. The E-Holder™ is used to load multiple gels on a robotic platform while other gels are running on the E-Base™.

E-Editor™ 2.02 Software

• The E-Editor™ 2.02 software allows you to quickly reconfigure digital images of E-Gel 48 or 96 gel results for analysis and documentation. The E-Editor™ 2.02 software can be downloaded for free from our Web site at www.lifetechnologies.com/egels.

Choosing a Gel for Your Application

To obtain the best results for your application, it is important to choose the correct agarose percentage and well format.

The table below lists the various types of gel and resolution for each gel type.

*Wells compatible for loading with a multichannel pipettor.

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 E-Gel^® With SYBR Safe^®
 
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 (figure below) is an easy-to-use, automated device specifically designed to simplify electrophoresis of single comb or double comb E-Gel^® agarose gels from Invitrogen. 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 (see figure below) 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^® Opener
 
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^® for excision of DNA fragments or for blotting.

• 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.

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 (E-Gel^® 1.2% with SYBR Safe™) or 700 ng per lane (E-Gel^® 2% with SYBR Safe™) of 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 15 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 into any empty wells.

Loading Buffer

• Use of loading buffer is optional.
• 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 50- to 200 fold diluted 10X BlueJuice™ Gel Loading Buffer or TrackIt™ Loading Buffer, 10% glycerol may be added.

High Salt Samples

Important: To obtain the best results, prepare your high salt samples as described below.
 
Samples containing > 20 mM NaCl or 5 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 2 µl 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^® with SYBR Safe™ agarose gels. Consider using Clear E-Gel^® agarose gels and post-staining with SYBR Safe™ DNA Gel Stain.
 

Introduction
 
After you have prepared your samples, you are ready to proceed with electrophoresis. Instructions are provided below to load and run E-Gel^® with SYBR Safe™ (which come as single comb gels) using the E-Gel^® PowerBase™ v.4.The total run times are 30–33 minutes.
 
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 adaptor 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^® with SYBR Safe™ agarose gels are designed for loading samples manually using either the One-Step Loading method or the Two-Step Loading method. The One-Step Loading method is routinely used to load E-Gel^® with SYBR Safe™ gels; use the Two-Step Loading method, if the One-Step Loading method produces fuzzy or indistinct bands, or the gel was removed from its plastic pouch for more than 15 minutes.
 
One-Step Loading Method

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^® with SYBR Safe™ 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 20 µl of sample per sample well.
3. Load 20 µl (500-700 ng) of the appropriate molecular weight markers.
4. Load 20 µl of water into any remaining empty wells.

 
Alternate Method:

Two-Step Loading

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^® with SYBR Safe™ 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 10 µl of deionized water into each well (including sample, molecular weight marker and empty wells). Do not premix with sample.
3. Load 10 µl of sample containing per sample well (10% glycerol may be added).
4. Load 10 µl (500-700 ng) of the appropriate molecular weight markers (10% glycerol may be added).
5. Load 10 µl of water into any remaining empty wells (10% glycerol may be added).

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 (see Figure 1).



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.

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.

• 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. 

 
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 (see Figure 1). 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.
 
Cleaning and Storage
 
After use, clean the E-Gel^® Opener with mild detergent and water to remove excess agarose 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.
 
Disposal of E-Gel^® with SYBR Safe™
 
SYBR Safe™ DNA gel stain shows no or very low mutagenic activity when tested by an independent, licensed testing laboratory, and this stain is not classified as hazardous waste under U.S. Federal regulations. As disposal regulations vary, please contact your safety office or local municipality for appropriate SYBR Safe disposal in your community.

E-Gel^® 1.2% with SYBR Safe™

An example of DNA samples run on an E-Gel^® 1.2% with SYBR Safe™ is shown below. Samples were loaded in a total volume of 20 µl and visualized on a standard 312 nm UV transilluminator. Photographs were taken using the MiniBis photo documentation system from DNR, and the SYBR Safe™ photographic filter using an exposure time of 1.8 sec.

E-Gel^® 2% with SYBR Safe™
 
An example of DNA samples run on an E-Gel^® 2% with SYBR Safe™ is shown below. Samples were loaded in a total volume of 20 µl and visualized on a standard 312 nm UV transilluminator. Photographs were taken using the MiniBis photo documentation system from DNR, and the SYBR Safe™ photographic filter using an exposure time of 1.8 sec.

Examples Using Different Imaging Methods
 
DNA samples run on an E-Gel^® 2% with SYBR Safe™ are shown below, recorded using different imaging methods. Samples were loaded in a total volume of 20 µl and visualized using the indicated transilluminator and filter. Photographs were taken using the MiniBis photo documentation system from DNR.


Standard 312 nm UV transilluminator
Long pass ethidium bromide filter

E-Gel^® with SYBR Safe™




Standard 312 nm UV transilluminator
SYBR Safe™ photographic filter

E-Gel^® with SYBR Safe™




Blue-light transilluminator
SYBR Safe™ photographic filter
 
E-Gel^® with SYBR™ Safe

The table below provides solutions to some problems that you may encounter with E-Gel® with SYBR Safe™ agarose gels.

Filter Selection Guide
 
Use the filter recommended with your instrument below to photograph E-Gel^® with SYBR Safe™. We have shown the most popular instruments; other instruments with an excitation source in the UV range or between 470–530 nm may also be used with the proper filter. Contact your instrument manufacturer for advice.