Documents & Support

Having everything at room temperature means not having to thaw reagents, thus saving time in gel casting.

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

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Similarly to ethidium bromide, SYBR Safe DNA Gel Stain runs in the opposite direction of the migrating DNA. This has no practical effect on the use of gels cast with SYBR Safe DNA Gel Stain, as only the very bottom of the gel will have a lower concentration of stain. This effect can be partially counteracted by staining the gel with SYBR Safe DNA Gel Stain after electrophoresis. Solutions of dye should not be added to the running buffer as this can cause breakdown of the dye at the electrodes and release toxic volatile compounds into the air.

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This could be due to:

*Debris in the well
*High salt in the sample (make sure that the salt concentration does not exceed 50-100 mM)
*Running buffer issue
*Gel casting error

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

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Possible cause:

*Carry-over contamination of sample from one well into neighboring wells due to loading error
*Contaminated running buffer
*Gel casting error: malformed wells

Remedy:

*Use a gel loading tip to load wells
*Reduce the sample volume
*Do not delay while loading wells
*Do not delay after the run, as proteins can diffuse horizontally; a full well left next to an empty well would eventually contaminate the empty well over time.

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

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After electrophoresis, the gel should be stained in a 0.5-1.0 µg/mL solution of ethidium bromide in deionized water for 15 to 60 min depending on the thickness of the gel. As an optional step to reduce background fluorescence, the gel can be destained in deionized water for 15 to 30 min. Alternatively, ethidium bromide may be added directly to the agarose prior to casting. Add ethidium bromide to melted agarose to a final concentration of 0.5 µg/mL. This has the advantage of reducing the amount of ethidium bromide waste. However, this procedure may reduce the migration rate of nucleic acids.

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Our NativePAGE gels do have a stacking gel. The stacking gel for these gels is a ~1 cm region at the top of the gel where the acrylamide percentage is low (4%) and constant. Below the stacking gel, the acrylamide percentage begins to increase in the gradient portion of the gel. However, the gel buffer is the same throughout the gel. So the stacking gel in NativePAGE gels is not the same as in the Laemmli system where the stacking gel has a different pH causing a decreased ion mobility for the trailing ions. Also the entire NativePAGE gel is cast in one continuous delivery due to which no demarcation line is seen between the resolving (or gradient) portion of the gel and the stacking gel.

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

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The SureCast Handcast System and reagents are available both as a package or as standalones.

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

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There are multiple factors that can result in PCR products that produce a smear of DNA or non-specific bands on an electrophoresis gel. Potential causes as well as recommendations for troubleshooting each are below:
1. Low annealing temperature - primers can bind off-target sequence if the annealing temperature is too low. To improve specific binding we recommend that you:
  • Increase the primer annealing temperature, up to a maximum of 68°C.
  • Set up a gradient PCR to determine the optimal annealing temperature for your primer pairs.
2. Sub-optimal cycling parameters - we recommend optimizing your PCR cycling conditions by trying the following:
  • Reduce the number of cycles in your PCR program.
  • Reduce the length of one or more of your cycle segments (denaturation, annealing, or extension).
3. Primers - issues with incorrect primer storage, poor primer quality, as well as sub-optimal primer design or concentration can negatively affect PCR amplification. To address primer issues we recommend the following:
  • Make aliquots of the primer stock and store primers at -20°C.
  • Make sure primers are not degraded.
  • If working with established primer pairs, check primer performance with a control template, under established PCR conditions.
  • Review primer design and if needed, design alternative primer pairs.
  • Determine the optimal primer concentration for the reaction. The final concentration range is generally 0.3-0.6 µM.
  • Make sure sense and anti-sense primers are present in equal concentrations.
  • For targets ≥15 kb, design multiple primer pairs to increase the chance of amplification.
4. DNA template is degraded or at the wrong concentration - degraded DNA template, as well as adding too much or too little of the DNA template can result in poor yield from your PCR. To troubleshoot this issue:
  • Test serial dilutions of your DNA template.
  • Check the quality and purity of your DNA.
5. Contamination with previous PCR product or other non-target template - to resolve poor amplification caused by contamination, we recommend that you:
  • Replace all of your reagents.
  • Use separate areas for PCR set-up and PCR product analysis.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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Our Zymogram gels do not contain the substrate (gelatin or casein) in the stacking gel although a small amount might mix in during casting of the gel.

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

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To help you choose the TBE polyacrylamide gel that will provide optimal separation results for your nucleic acid fragment, we have designed gel migration charts for each gel type offered. Please check the TBE gel migration charts on page 74 of the Novex Pre-Cast Gel Electrophoresis Guide (https://tools.thermofisher.com/content/sfs/manuals/electrophoresisguide_man.pdf).

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

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We do offer disposable, empty plastic cassettes and combs to provide the option of pouring your own Mini or Midi gels for use with the XCell SureLock Mini-Cell, Mini Gel Tank, or XCell4 SureLock Midi-Cell, respectively. All cassettes are sealed on three sides to prevent leaking. Since the cassettes are pre-sealed with the slot pre-taped, a bulky casting stand is not necessary. The empty cassettes and combs can be found here (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/power-supplies-protein-electrophoresis.html#staining).

- The Empty Mini Gel Cassettes are available in two thicknesses (1.0 mm and 1.5 mm) and can be used with Invitrogen Combs that are available in two thicknesses (1.0 mm and 1.5 mm), and in 7 types (1 well, 2 well, 5 well, 10 well, 12 well, 15 well, and 2D well).
- The Bolt Empty Mini Gel Cassettes are available in 1.0 mm thickness and can be used with Bolt 10- or 12-well combs.
- The Empty Midi Gel Cassettes are available in 1.0 mm thickness and can be used with three Invitrogen Comb types (12 + 2 wells, 20 wells, and 26 wells).

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

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The NativePAGE Invitrogen Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analyzing native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessing purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow.

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

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Invitrogen Tris-Glycine polyacrylamide gel chemistry is based on the Laemmli system with minor modifications for maximum performance in the pre-cast format. These gels do not contain SDS and can therefore be used to accurately separate both native and denatured proteins by using the appropriate running buffer. Invitrogen Tris-Glycine Gels provide reproducible separation of a wide range of proteins into well resolved bands.

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

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Here are possible causes and solutions:

- High voltage application is set to run on a very low current. DISABLE No Load alarm on the Running Screen (see manual for details), for example when performing an isoelectric focusing application.
- Electrophoresis leads are not connected to the power supply or to the electrophoresis unit(s), or there is a broken circuit in the electrophoresis cell. Check the connections to the power supply and on your electrophoresis cell to make sure the connection is intact; check condition of wires in electrophoresis unit. Close the circuit by reconnecting the cables. Press Stop/Start to restart the run.
- High resistance due to tape left on a pre-cast gel, incorrect buffer concentration, or incorrect buffer volumes in the electrophoresis cell. Correct the condition by making sure the tape is removed from the pre-cast gel, buffers are prepared correctly, and the recommended volume of buffer is sadded to the electrophoresis unit.

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

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