agarose gel electrophoresis for short oligonucleotides


Question

Maximum resolution on agarose gel

I am looking for alternative splice variants in a cDNA preparation. The fragment size is around 1000bp. I want to know what is the maximum resolution I can expect from agarose gel electrophoresis? What parameters should I manipulate to get the best resolution?

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ALL ANSWERS (20)

  • Gal Haimovich · Albert Einstein College of Medicine
    I think you can get to a resolution of ~50bp or even less, but you’ll need to have at least 1.5-2% gel (the higher the concentration, the better resolution, but the DNA runs slower), and run it for a long time. (Use a large gel, not minigel, otherwise the fragments will leave the gel before you get to see any separation).
  • Kiev Blasier · University of Virginia
    Varying the agarose concentration is going to be your best way for getting optimal resolution. (Higher concentration = smaller pore size = better separation) Some companies even offer what they call high resolution agarose gels, though I have never used them. In my experience the smallest I’ve been able to really satisfactorily separate is 50 bp. Hope this helps.
  • Lekha Margassery · University College Cork
    I would suggest using 1.2% agarose gel (mini gel) and applying 50-100V for 1-2 hrs.
  • Juliana Americo · Federal University of Rio de Janeiro
    Hi Chandima. The first thing you should adjust is the agarose concentration. For fragments around 1 Kb I would suggest you to use at least 2% of agarose. But what is the size difference you expect among these variants?
    Another thing is that I usually have better results using high resolution agarose. The manufacturer says that with high resolution agarose one can separate 2 fragments differing only in 2% in their sizes. But I never tested it with such a small difference.
  • Eduardo Fernandez-Rebollo · IDIBAPS August Pi i Sunyer Biomedical Research Institute
    Dear Chandima, with the regular agarose you could get a resolution of around 20bp with a 4% gel, but if you expect that the alternative splice variants are going to differ in less than 20bp I recommend you to use the MetaPhor agarose 2-4% (Lonza) that gets a really high resolution. Good luck
  • Elijah Magrane · McGill University
    All of the above recommendations not withstanding I found that one of the best ways to increase resolution of an agarose gel is to cast a thin gel.

    Of course, an alternative method for achieving maximum resolution would be just to run acrylamide gel (I have resolved 10bp). Also don’t forget to account for the amount of mass you’re loading. Typically in an agarose gel (depending if your staining with EB or SG) you can only resolve ng of nucleic acids

  • Koen Venken · Baylor College of Medicine
    I use 2.5% to see a difference of 40bp well. But the fragments are 200 to 300 bp. Since yours is 1kb, I suggest to run a long gel. If different primers are used for the different splice forms, you can increase the length of the primers for the longer one with “dummy” tail sequence, just to increase the length of the amplicon artificially.
  • Kamal Sharma · Czech University of Life Sciences Prague
    Hi Chandima,
    Use 2% high resolution agarose gel (sigma). Handle it with precautions as it is very delicate than the routine agarose gel. If it is not available to you at the moment use 2% agarose gel (routine use). Run it on a large gel for long time at 60 volt.
    Good luck
  • Ibrahim Thabet · Zagazig University
    I think that 1.5 % molecular biology grade agarose would be the best …. beside decreasing the voltage of electrophoresis for slow and so good separation of bands …
  • Zahida Pervaiz · University of Pittsburgh
    fresh TBE buffer for electrophoresis, use metaphore agarose ,its high resolution agarose 3% is enough for separation, and after making gel put the gel tray for 10 min at low temperature in order to make it fully solid. You will get best results
  • Oneida Morales · University of the Valley of Guatemala
    Hi Chandima maybe you could try with SeaKem LE Agarose the purpose for this agarose is Analytical electrophoresis of DNA and RNA ≥1,000 bp.
    It really works very well!!
    Look for more . Here are some information
    http://www.takara.co.kr/file/manual/pdf/50000.pdf
    http://www.takara.co.kr/file/manual/pdf/50150.pdf
  • Lucy Robinson · Louisiana State University Health Sciences Center Shreveport
    For a 1 kb fragment, resolution is pretty good at standard 1% agarose concentration. Like a couple of other respondents, I would go a little higher- 1.2% and 1.5% were suggested and either may work better than the other. If you have enough sample, you could try several different concentrations. There were a couple of other suggestions besides agarose brand and concentration. In my experience, running the gel slower than usual could help your resolution, although running too slowly could actually decrease effective resolution. Good agarose and fresh buffer are always useful. Running a relatively thin gel could help effective resolution by decreasing the out of focus light when you image, but it would be even better to ensure that you do not overload, as you stand a better chance of resolving bands when the bands are very thin. Have you tried electrophoresis of this mix yet? Maybe you should start with your standard conditions (or improved standard conditions- good agarose, fresh buffer, and voltage to result in not more than 50 mA) and change one parameter at a time to see what works best.
  • Belma Kalamujic · Institute for Genetic Engineering and Biotechnology
    I agree with Lucy. Even though the rule says “the greater the percentage of gel, the better the resolution” it usually works for fragments smaller than the one you’re dealing with. Freshness of the buffer is of utmost importance since you’ll need to run your gel longer. And loading volume will also affect your ability to differ between two fragments. Even though you do most of other things right, you still won’t be able to differentiate two fragments of just dozen of bp difference if your bands are too thick.
  • Sara Ohadi · University of Melbourne
    I used Metaphor agarose, it has a high resolution with less than 10bp. But it is very delicate. I usually made a 3% agarose. Also you if you want to use it, it would better to set the voltage around 90. This would be take longer, let’s say about 2 hour, for band separation.
  • Deleted
    Hi, i guess u dont need to switch to any other special agarose. You can use 1% agarose gel and 1kb DNA marker,stain with EtBr. I think your DNA size is not strange..this size is normally reported in many places and experiments. Well try, i guess you will see clear sharp band if template concentration and PCR conditions are good.
  • Ishtiaq Khan · University of Karachi
    Use 1.5% high resolution gel and 1-2V/cm voltage. Voltage plays important role in this case.
  • Pandit Ramesh · ARIBAS
    Use 1.5% gel. And one thing that i have do during my work is that i use cool (10-15 degree Celsius) 1% TBE Buffer and its gives good resolution.
  • Lenin Godoy · Ponce School of Medicine and Health Sciences
    The resolution will depends on voltage and running time. For better resolution run your gel slowly. Obviously, the concentration of your DNA sample and the staining agent (SYBR or EtBr) are very important. SYBR is more sensitive than EtBr and less toxic. I work with TBE and TAE and I see no difference in resolution.
  • Andrew Jenkins · Telemark University College
    You can expect a resolution of 5% of the fragment size or better.
    Don’t add stain to the gel during the run as it is difficult to maintain a constant concentration.
    Don’t make the gel too thick, but don’t go to extremes of thinness either. Don’t let it get too warm – buffer circulation is a good idea, but it’s a hassle.
    You may like to try a trick I have done by accident a few times: run the gel backwards for 15 minutes, then reverse the electrodes. This works as a kind of poor mans FIGE (field inversion gel electrophoresis). The few times I’ve done this the results have been great.
    I agree with the others about concentration. 1 – 1.5%. 3% is too high for a 1000 bp fragment.
  • Chandima Ariyarathna · University of Western Australia
    Hi,
    Thanks everybody for the comments. They were really helpful. I manage to get a good resolution at 1.5% agarose and running slowly at 80V in a very thin gel (>4mm). The fragments separated enough to do gel purification. I got them cut from the gel and cloned. Great help, THANKS
    Cheer
    Chanidma

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