I am getting poor resolution or see smearing of bands when using my E-Gel™ EX Agarose Gels. Why is this?

Product FAQ

Answer

Here are some possibilities and suggestions to resolve the problem:

  • The sample is overloaded. Do not load more than the recommended amount.
  • A high salt concentration. Dilute the samples.
  • The E-Gel™ EX Agarose Gels were prerun; this is not recommended for these gels.
  • A very low volume of sample was loaded or the sample was not loaded properly.
  • Bubbles may have been introduced while loading the samples. Bubbles will cause band distortion. Load the recommended sample volume based on the gel type and loading method. For proper band separation, we recommend keeping sample volumes uniform. Load deionized water or TE into any empty wells, and avoid introducing bubbles.
  • The gel was not electrophoresed immediately after sample loading. Run your sample within 1 minute of loading it.
  • The E-Gel™ Agarose Gel may have been used beyond its expiration date. Check the expiration date.
  • A longer electrophoresis run time or high current was used during the run. Longer run times cause an increase in the current, resulting in poor band migration or a melted gel. Do not run the gel longer than the recommended time for each gel type.

    Answer Id: E8023

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  • I am trying to run my E-Gel™ software on my Mac™ computer, but it’s not working. What should I do?

    Product FAQ

    Answer

    We do not currently offer software that is compatible with Mac™ computers. Please install the software on a 32-bit or 64-bit PC running either a Windows™ XP Professional or Windows™ 7 Professional operating system.

    Answer Id: E8024

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    I am getting the error message: Deletfile: code 5. Access is denied when running my E-Gel™ software. I have tried this on three computers. What can I do?

    Product FAQ

    Answer

    This error can occur if there is other software running in the background, like an Internet Explorer™ browser, or if there is an antivirus program running that is limiting his ability to access some files. To fix this error, try to:

  • Turn off Internet Explorer™ or any other program running during the install.
  • Inactivate the antivirus program (or temporarily uninstall it), install the E-Gel™ Imager software, then reactivate or reinstall the antivirus program.
  • When the error comes up, select "Ignore" to allow the installation to continue instead of "Retry" or "Abort".

    Answer Id: E8025

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  • The E-Gel™ Imager light does not stay illuminated long enough to get a good image. What can I do?

    Product FAQ

    Answer

    Depress the activation button for at least 2 seconds, and the imager will transilluminate for 5 minutes rather than 30 seconds.

    Answer Id: E8026

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    Do you have a protocol for ethidium bromide staining in agarose gels?

    Product FAQ

    Answer

    For use in agarose gels:

  • Add ethidium bromide to melted agarose to a final concentration of 0.5 μg/mL. Do not melt agarose already containing ethidium bromide. For staining agarose gels after electrophoresis:
  • You can stain gels that have been run in the absence of ethidium bromide by covering the gel in 0.5 μg/mL ethidium bromide in water and gently agitating for 10 to 30 minutes.
  • If necessary, gels can be destained by shaking in H2O for an additional 30 minutes. To stain RNA gels, you should minimize staining time and definitely include a destaining period.

    Answer Id: E8043

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  • The base indicator light is on but it is not transilluminating when using the E-Gel™ Imager.

    Product FAQ

    Answer

    1) Check that the camera hood or activator key is in place to make sure the base is not deactivated.
    2) Light bases will "time out," but the power indicator light may stay illuminated. Turn the base off, then back on.

    Answer Id: E8027

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    What are the differences between all the SYBR™ dyes?

    Product FAQ

    Answer

    SYBR™ Green I is used for staining dsDNA and ssDNA. SYBR™ Green II is used for RNA. These stains are preferred if you will use gel scanning devices to image the gel. In most applications, SYBR™ Gold is a bit more sensitive than either SYBR™ Green I or SYBR™ Green II (because it is a better general-purpose stain), and it is preferred for documentation with black and white photography. SYBR™ Safe DNA Gel Stain is a reduced mutagenicity formula designed for use with blue light systems. It is somewhat less sensitive than the other forms.

    Answer Id: E8044

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    What is the storage temperature for the Krypton Protein Stain and how stable is it?

    Product FAQ

    Answer

    Store Krypton Protein Stain at 4 degrees C; although it is stable enough for ambient shipping. The 10X solution is under warranty for one year from the date of purchase if handled and stored properly. The Working Solution (1X) is stable at 4 degrees C for 1-2 weeks.

    Answer Id: E8380

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    My TBE buffer has precipitated out of solution. What can I do?

    Product FAQ

    Answer

    If a slight turbidity develops, the fine precipitate can be dissolved by autoclaving for 5 minutes at 110°C. Do not autoclave in the container supplied. This treatment has no deleterious effect on the buffering properties of TBE.

    Answer Id: E8028

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    How do these dyes bind to DNA?

    Product FAQ

    Answer

    The binding mode of the SYTO™ nucleic acid stains is unknown. However, the behavior of these and related nucleic acid dyes suggests the following binding properties:

    1.They appear to contact the solvent (suggested by sensitivity to salt, divalent cations, and in particular, SDS) and thus are likely to have contacts in the grooves.
    2.All SYTO™ dyes appear to show some base selectivity and are thus likely to have minor groove contacts.
    3.They can be removed from nucleic acid via ethanol precipitation; this characteristic is not shared by ethidium bromide and other intercalators. Likewise, the dyes are not removed from nucleic acid via butanol or chloroform extraction. These extraction methods do remove ethidium bromide from nucleic acid.
    4.SYTO™ binding is not affected by nonionic detergents.
    5.SYTO™ dyes are not quenched by BrdU, so they do not bind nucleic acids in precisely the same way as Hoechst 33342 and DAPI ((4′,6-diamidino-2-phenylindole).
    6.SYBR™ Green I has shown little mutagenicity on frameshift indicator strains, indicating that it isn't likely to strongly intercalate.

    Answer Id: E8045

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    If the Krypton Protein Stain is cloudy, can I still use it?

    Product FAQ

    Answer

    Yes. The solution will be cloudy immediately upon dilution to 1X, especially if it is cold. The cloudiness should dissipate with mixing.

    Answer Id: E8381

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    I am getting high background and smeared bands on my TBE-urea gels. What should I do?

    Product FAQ

    Answer

    Here are some suggestions for your experiments:

  • The RNA samples may have been degraded by RNases. Use standard precautions to prevent contamination.
  • Make sure samples are heated for 3 minutes at 70°C just prior to loading. If this is not done, the oligonucleotides will not be fully denatured, which may result in a smeared background.
  • Be sure to vigorously flush urea out of sample wells just prior to loading the sample. Urea will continually seep from the gel into the well. Urea is very dense and will force the sample into a ball.
  • A sample volume over 10 μL may result in smearing. This volume is less than may be used with agarose gels.
  • If ultrapure water (18 milliohms) is not used, smeared bands and high background may result.
  • Check to make sure the running and sample buffers have been prepared and diluted correctly.
  • Make sure to run the bromophenol blue until it reaches the slot. Stopping the run short can result in less than optimal results.

    Answer Id: E8029

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  • What is the difference between the TrackIt™ DNA Ladders and other DNA ladders?

    Product FAQ

    Answer

    TrackIt™ DNA Ladders contain two tracking dyes in the sample buffers, which serve as visual markers for tracking electrophoresis progress through the gel, and also to indicate when maximum resolution is achieved. The tracking dyes should not obscure your visualization of DNA bands in the ladder, as the dyes run outside the limits of most DNA bands in the ladder.

    TrackIt™ Cyan/Orange Loading Buffer is formulated with unique tracking dyes, Xylene Cyanol FF and Orange G. We recommend TrackIt™ Cyan/Orange Loading Buffer for DNA fragments between 10 bp and 1 kb.

    The TrackIt™ Cyan/Yellow Loading Buffer is formulated with unique tracking dyes, Xylene Cyanol FF and Tartrazine. We recommend TrackIt™ Cyan/Yellow Loading Buffer for DNA fragments between 100 bp and 10 kb. The molecular weights are Xylene Cyanol FF, 638.6; Orange G, 452.4; Tartrazine, 534.4.

    Note: The TrackIt™ DNA Ladders are not recommended for use with polyacrylamide gels and are not designed for quantitation.

    Answer Id: E8063

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    How should I dispose of SYBR™ DNA Gel Stain?

    Product FAQ

    Answer

    Disposal regulations vary. Please contact your safety office or local municipality for disposal guidelines.

    Answer Id: E8046

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    I think I’ve added too much virus. How important is the MOI? What happens if too much virus is used?

    Product FAQ

    Answer

    An MOI of 5-10 is typically used. If too much virus is added, unfortunately the cells die too soon and the protein expression level goes down.

    Answer Id: E9466

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