Manual / Product Insert

User Guide: Non-denaturing Agarose Gel Electrophoresis

Version: Jan. 2015
Catalog #

Product FAQ

At what voltage should I run an agarose gel prepared from TopVision Agarose Tablets?

Answer

The electrophoresis conditions do not depend on the form of delivery of agarose. Whether it is loose powder or tablets, the material has the same physicochemical conditions. In general, 4-10 volts/cm (cm is determined by measuring the interelectrode distance, not the gel length) are recommended when running agarose gels under normal horizontal electrophoretic conditions.

Answer Id: E8721

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Citations & References

Molecular stretching of long DNA in agarose gel using alternating current electric fields.

  • Authors: Kaji N, Ueda M, Baba Y
  • Journal: Biophys J (2002) 82:335-344
  • PubMed ID: 11751320
Catalog #

Product FAQ

What loading buffer should I use for my E-Gel Agarose Gels?

Answer

Loading buffer is optional. Samples can be loaded directly into the wells if no buffer is used, or you can dilute them with deionized water or TE buffer. If you want to use a loading buffer, please see the recipes below:

E-Gel Agarose Gels (including EX)
10 mM Tris-HCl, pH 7.5
1 mM EDTA
0.005% bromophenol blue
0.005% xylene cyanol FF
E-Gel CloneWell and E-Gel SizeSelect gels
10 mM Tris-HCl, pH 7.5
1 mM EDTA

Alternatively, you can use 10X BlueJuice Gel Loading Buffer or TrackIt Loading Buffer. Dilute this buffer 50- to 200-fold to obtain optimal results with E-Gel Agarose Gels.

Answer Id: E7985

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Product FAQ

I am seeing multiple DNA bands after running a single vector on my agarose gel. Why is this?

Answer

The conformation of plasmid DNA will affect its mobility on a gel. Plasmid DNA can be supercoiled (native 3D conformation), nicked circular (nick in one DNA strand) or linear (nicks in both DNA strands). You may see all three forms on an agarose gel. Supercoiled DNA runs the fastest and will show up lowest on the gel, linear DNA runs in the middle of the gel, and nicked circular DNA shows up at the top of the gel, since it migrates the slowest.

Answer Id: E8015

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Citations & References

Comparison of ethidium bromide and 4', 6'-diamidino-2-phenylindole as quantitative fluorescent stains for DNA in agarose gels

  • Authors: Nairn, R.S., et al.
  • Journal: J Biochem Biophys Meth (1982) 6:95-103

Product FAQ

I’m running an agarose gel with TAE or TBE. Can I use your gel extraction kits?

Answer

Yes, both TAE and TBE agarose gels are compatible with our gel extraction kits.

Answer Id: E7710

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Citations & References

SYBR Green I staining of pulsed field agarose gels is a sensitive and inexpensive way of quantitating DNA double-strand breaks in mammalian cells.

  • Authors: Kiltie AE, Ryan AJ
  • Journal: Nucleic Acids Res (1997) 25:2945-2946
  • PubMed ID: 9207049

Citations & References

Automated agarose gel electrophoresis of dsDNA fragments on a commercial DNA sequencer.

  • Authors: Khandurina J, Legg E, Wang X, Guttman A
  • Journal: Biotechniques (2002) 33:1008-1008
Catalog #

Product FAQ

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

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).

Answer Id: E7958

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Manual / Product Insert

ReadyPouch Agarose Gel Protocol

Version: Rev. A (8 April 2014)
Catalog #
  • A25647(Discontinued)
  • A25648(Discontinued)

Product FAQ

Is it important to include water in the second row of wells for the E-Gel SizeSelect Agarose Gels and E-Gel CloneWell Agarose Gels?

Answer

Yes, please ensure that the second row is filled with sterile water prior to running your band of interest into the collection well. Please note that the refill volume may vary between wells. Do not overfill.

Answer Id: E7967

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Product FAQ

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

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.

Answer Id: E7986

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Product FAQ

My sample is leaking from the wells when running my E-Gel Agarose Gels. What happened?

Answer

Please ensure that you have not overloaded the well and that the wells were not damaged during comb removal.

Answer Id: E8018

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