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Do you have a protocol for transferring RNA from my E-Gel EX Agarose Gels for Northern blotting? Product FAQ

Answer

Here is a suggested protocol:

- Prewet a nylon membrane suitable for use with RNA using 5X SSC buffer.
- Using the E-Gel Opener, 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.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

Answer Id: E7986

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Quick Ref: E-Gel EX Agarose Gel - Japanese Manual / Product Insert

  • Version: EGel EX Agarose Gel_QRC_J1_6Aug2009.doc
Catalog #
  • G401001
  • G401004
  • G402001(Discontinued)
  • G402002(Discontinued)
  • G6511ST(Discontinued)
  • G6512ST(Discontinued)
  • G4010-02

Quick Ref: E-Gel® EX Agarose Gel Manual / Product Insert

  • Version: Rev date: 6 August 2009
Catalog #
  • G401001
  • G401004
  • G402001(Discontinued)
  • G6512STEU(Discontinued)
  • G402002(Discontinued)
  • G6511ST(Discontinued)
  • G6511STUK(Discontinued)
  • G6512STUK(Discontinued)
  • G6512ST(Discontinued)
  • G6511STEU(Discontinued)
  • G4010-02

What is the special feature of E-Gel EX agarose gels? Product FAQ

Answer

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 have a sensitivity that is 5X greater than gels using ethidium bromide.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

Answer Id: E7964

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I am not seeing any current when I try to run my E-Gel EX agarose gels. Why is this? Product FAQ

Answer

Here are some common reasons why your gel is not running properly:

- Copper contacts in the base are damaged due to improper use. Make sure the copper contacts in the base are intact.
- An expired or defective gel cassette was used.
- The E-Gel EX cassette was not inserted properly into a base.
- An incorrect adaptor was used.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

Answer Id: E8022

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What are the dimensions of a single comb 12 well E-Gel agarose gel? Product FAQ

Answer

The dimensions of a single comb 12 well E-Gel agarose gel are as follows: The cassette itself is 8 cm by 10 cm and 0.6 cm thick. The workable gel area is 7.5 cm by 5.8 cm (from well to bottom). The thickness of the gel is estimated to be 0.4 cm. There are 12 wells and each well is 4 mm wide. The space between the wells is 1 mm.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

Answer Id: E3367

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Can I use double comb E-Gel EX agarose gels or E-Gel agarose gels with SYBR Safe stain for RNA analysis? Product FAQ

Answer

E-Gel EX Double Comb agarose gels and E-Gel Double Comb agarose gels with SYBR Safe stain have not been tested for use in RNA applications. We recommend using E-Gel EX Single Comb agarose gels with 11 wells for RNA analysis.

Answer Id: E18346

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I have an E-Gel Simple Runner Electrophoresis Device in my lab, are the new 11-well and 22-well E-Gel agarose gels compatible with it? Product FAQ

Answer

Due to the differences in dimensions, the new E-Gel 11-well and 22-well agarose gel formats are not compatible with the E-Gel Simple Runner Electrophoresis Device.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

Answer Id: E18351

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What is the function of the E-Editor 2.0 software? Product FAQ

Answer

The E-Editor 2.0 software allows you to quickly reconfigure digital images of E-Gel 48, E-Gel 96, E-PAGE 48 and E-PAGE 96 gel results for analysis and documentation. You can capture an image of the gel and then use the E-Editor 2.0 software to:
-Align and arrange the lanes in the image to any 48, 96, or 384 image
-Save the reconfigured image for further analysis
-Copy and paste selected lanes or the entire image into other applications for printing, saving, e-mailing, and/or publishing on the Web.

The E-Editor 2.0 does not take perform densitometry analysis from your gel images. The E-Editor 2.0 can be downloaded for free.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Answer Id: E4868

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How much DNA can I load for my E-Gel Agarose Gels? Product FAQ

Answer

To determine the amount of DNA to load per well for your specific E-Gel agarose gel, please refer to the table on page 14 of the E-Gel Power Snap Electrophoresis System user guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017050_egel_powersnapsystem_UG.pdf).

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

Answer Id: E7981

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How can I get better separation of my bands? Product FAQ

Answer

First check the percentage of your agarose gel. A higher percentage will help you to resolve smaller molecular weights while a lower percentage will help you to resolve larger molecular weights.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

Answer Id: E8010

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What does intact RNA look like when run on an agarose gel? Product FAQ

Answer

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, visit this website (https://www.thermofisher.com/us/en/home/references/protocols/nucleic-acid-purification-and-analysis/rna-protocol/agarose-gel-electrophoresis-of-rna.html).

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

Answer Id: E7958

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How can I perform RNA electrophoresis? Product FAQ

Answer

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, 10 mM 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.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

Answer Id: E7957

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Should I use agarose or polyacrylamide gels for DNA electrophoresis? Product FAQ

Answer

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 Invitrogen TBE polyacrylamide gels and UltraPure reagents.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

Answer Id: E7956

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Can I dispose of E-Gel EX agarose gels the same way as E-Gel agarose gels with SYBR Safe DNA Gel Stain? Product FAQ

Answer

No. Standard safety and hazardous waste disposal procedures should be followed when handling E-Gel EX agarose gels.

Answer Id: E18344

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