Find content
Not Telling
I am interested in hearing what others are using.
Our lab is required to treat used EtBr (in gels and running buffer) as hazardous waste. This is expensive ($100 per pickup, 25 L pail of used gels), time consuming, paperwork, hastle, etc. To remove the EtBr from the used running buffer we stir overnight with "tea bags" from Amresco. Afterward we pour the running buffer down the sink. After 50 uses we discard the used tea bags with our used gel waste.
I've tested EZ Vision (Amresco), Sybrsafe (Invitrogen) and GelRed (Biotium).
I'm not plugging any product here. I only care about what works without any bias as to where I buy it from. We only have the camera filter for EtBr. EZ Vision and Sybrsafe require a the Sybrgreen camera filter ($900 - yikes!). However, GelRed works well with the EtBr camera filter.
Although Sybersafe and EZ-Vision appear to work well, the long exposure required (EtBr camera filter) causes too much background fluorescence. I would like to test the EZ-Vision with a Sybersafe camera filter because the safety data on this product appears to be very thorough. I think EZ-Vision can actually be discarded as a truly non-hazardous substance in regular garbage (double bag the used gels and pour used running buffer down sink with running tap water).
For time being I opted to pursue GelRed further because it uses EtBr filter.
At first I tried using GelRed in the gel but the PCR bands were too strong with smeary frown. I suspect the problem of heavy smeary frown may be due to overloading the gel. However, the smeary frown was still a problem, even after I tested serial dilutions of the DNA and different concentrations of GelRed in the gel.
In this whole experiment I tested a variety of PCRs between 100 bp and 1,000 bp.
I decided to try instead using GelRed in the loading buffer. I used gels and running buffer without any dye. This appears to work much better.
I tried the following amounts of GelRed in loading buffer:
1ul/100ul, 1ul/200ul, 1ul/500ul, 1ul/1ml, 1ul/2ml, 1ul/5ml
I added 5ul of GelRed loading buffers to 25 ul PCRs and loaded 10 ul into gel.
The gel and running buffer (1x TAE) do not contain any dye.
1/100 and 1/200 GelRed in loading buffer gives too strong staining for most of our PCRs, which requires us to titrate how much PCR to load on gel. It also gives some background fluorescence.
1/500, 1/1,000 and 1/2,000 GelRed in loading buffer appear to give optimal staining for most of our PCRs, which does not require us to titrate how much PCR to load on gel.
Although 1/5,000 GelRed in loading buffer works, it may give too weak staining for some of our PCRs.
See photo.
Several years ago we stopped using agarose gels and instead use agarose-composite polysaccharide gels. These gels are stronger, more flexible, more clear (less background fluorescence) and have wider separation range than agarose. At first we used Visigel from Stratagene, but they discontinued the product in 2007. They kindly gave me the recipe. If anyone is interested I will post my recipes. It consists of 0.7% agarose and 1% clarified high molecular polysaccharide from Locust Bean Gum (aka, carobe bean). As far as I'm aware the only folk that make cHMW-LGB is Diversified Biotech, (Agarmate, AGM-200).
12-Aug 15 GelRed in loading buffer.jpg 52.12K
155 downloadsIf anyone is interested I will post my recipes.
