separation problem with GelRed

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    • #11716
      apolll
      Participant

      Hello!

      We recently switched from ethidium bromide to Gelred in our lab. (We always prestain gels – poststaining is no option because its too inconvenient and timeconsuming – using 1xTAE buffer).
      Our DNA samples and the ladders aren’t separated as sharply as it was when we used ethidium bromide. I already have some ideas what to try to improve the separation (using TBE buffer, using 0.5x buffer, applying lower voltage, etc.)
      Since I optimized several methods in the last months, I am really tired of it and I just wanted to ask you if you had the same problem and if you have found a solution for that or if you have any advice on what to test (first)?

      Thank you for your help!

    • #92565
      canalon
      Participant

      I am a bit surprised here, you say that post satining is too time consuming but you are ready to slow down your gels. It takes 10 minutes for staining, and about as much if you want to destain, which should not even be a problem with gelred. So why not post stain?
      If this is really not an option, lowering voltage is usually a good start. And re-using buffer less.

    • #97843
      weihan87
      Participant

      hi apolll,

      Regarding the separation problem with GelRed, i have encounter the same problem.
      i agree with you that GelRed give better AQ result, yet the only problem is the seperation.
      Do you have any solutions of advises for this case?
      Thanks

    • #98113
      buthercup
      Participant

      Hi!

      I’ve been using GelRed for a while, after using EtBR extensivelly, and I didn’t notice any change or problem with the separation of the bands… what gel % are you using?

      Im my lab, we usually use 0.8% agarose, TAE1X to disolve agarose, but 0.5X as running buffer, 100V. Adding the dye to the agarose prior to polymerisation is another option to the addition of the dye to the buffer. I’ve always had nice staining doing this way.

      Good luck

    • #98222
      weihan87
      Participant

      Hi there,
      after few tests, the similar problem still exist, especially for the ladder. my lab uses both 1% and 2% gel. 1x TAE for dissolving and also running. 100v for 50mins. I have tried using low voltage and higher voltage as well, yet the output still the same.
      Any suggestion or advise?

      Thank you

    • #100750
      cookydee
      Participant

      Hi there

      May I suggest that you try SYBR safe from Invitrogen (Molecular Probes). It functions similarly to Ethidium bromide by binding directly to the nucleic acids. I know Gel red functions differently.

      SYBR safe is also a non-toxic and non-carcinogenic alternative to Ethidium Bromide, which is microwavable with the agarose. The brightness of the bands was almost double that of ethidium.

      Good luck! I hope my suggestion is not too late for you.

    • #100767
      buthercup
      Participant

      Hi,
      I tried also SYBR safe and I’m happier with GelRed…,
      any way, I really encourage you to change the concentration fo your running buffer from 1X to 0.5X.

    • #111572
      citroenboom
      Participant

      We try this 0.5 x buffer protocol on the moment.
      Mixing (homogenizing) of the marker in the well also improved results.
      When done I will try to put a link to the gel pictures.

    • #111573
      citroenboom
      Participant
      quote weihan87:

      Hi there,
      after few tests, the similar problem still exist, especially for the ladder. my lab uses both 1% and 2% gel. 1x TAE for dissolving and also running. 100v for 50mins. I have tried using low voltage and higher voltage as well, yet the output still the same.
      Any suggestion or advise?

      Thank you

      Voltage didn’t make a difference. We have a student working on it.

    • #111576
      citroenboom
      Participant

      Just saw the gels. Type of marker makes a difference. If bands are close, the gel is easily overloaded. Overall, 0.5x buffer does not help. GelRed is out of here I think. Markers keep looking bad …

    • #112810
      annakarinring
      Participant

      I got the tip from another lab to dilute the DNA ladder and now it looks so much better, nice separation. We have diluted the ladder 1:10. Good for the lab-economy as well! Maybe one could go further even, and also dilute the markers- have not tried this yet. When I looked around I found that they acctually recommend dilution in their gelred FAQ http://www.biotium.com/product/product_ … lgreen.asp

    • #112973
      citroenboom
      Participant

      Sadly I did not help us out. In our lab often we want to check early if we have good product/restriction pattern. And with GelRed gels have to run longer to get nice bands.

      But it helps indeed if you run longer. Then result can be nice.

    • #115572
      Biotium
      Participant

      I know this is a very old thread, but we just wanted to chime in with an official reply and answers to your questions.

      GelRed is designed to be a large dye to increase its safety. Therefore, its large size may affect DNA migration in prestain gels, leading to some separation problems.

      Here are some solutions to improve band separation:

      1. Reduce the amount of loaded DNA. Overloading of DNA often results in band smearing. The recommended amount for ladders and samples of known concentration is 50-200 ng/lane. If the concentration is unknown, try loading one half or one third of the usual amount of DNA.
      2. Reduce the amount of GelRed. Try using 0.5X instead of 1X final concentration of GelRed.
      3. Pour a lower percentage of agarose gel. Higher molecular weight DNA separates better with a lower percentage gel.
      4. Change the running buffer. TBE buffer has a higher buffering capacity than TAE buffer.
      5. Try using the post-staining protocol if possible, especially if more than the recommended amount of DNA is required.

      For other questions regarding GelRed, check out Biotium’s FAQ
      http://biotium.com/support/faqs/faq-gel … el-stains/

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