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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!
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.
Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
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?
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.
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?
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.
Voltage didn't make a difference. We have a student working on it.
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
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.
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