Acrylamide gels blurring DNA - (Nov/08/2007 )
I've been running 10% PAGE, which have been working until recently. Now instead of giving distinct bands the DNA runs in a blurry smear, which is often too diffuse to even visualize. The gels are 10cmx10cm, and I'm trying to resolve bands around 200-300 bp with 4 bp resolution, so I'm running them for about 8 hours. I am cooling the gels, and I tried replacing AMPS (which seemed to partially help). I'm also using pretty new TEMED. The gels seem to be polymerizing properly (at least they're solid to the touch after removal from glass plates). What are the symptoms of old acrylamide? Anything else that could cause this? I've been racking my mind for 2 weeks. Many many thanks.
1. You may check the ratio of your acrylamide/Bis-acrylamide and your acrylamide &Bis-acrylamide may be out of date.
2. You may try to repalce your electrophoresis buffer，The buffer can affect the electrophoresis effect.[font="Times New Roman"][/font]
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2. You may try to repalce your electrophoresis buffer，The buffer can affect the electrophoresis effect.[font="Times New Roman"][/font]
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What ratio of acryl/bis should I be going for?
2. You may try to repalce your electrophoresis buffer，The buffer can affect the electrophoresis effect.[font="Times New Roman"][/font]
[size="4"][/size][color="#0000FF"][/color]
What ratio of acryl/bis should I be going for?
19/1 (common for sequencing gels), 29/1, or 37.5/1 (common for protein gels). what are you using?
you can try fresh acrylamide.
sounds as though you are seeing incomplete polymerization. you replaced aps but you say that your temed is nearly fresh, could it have been contaminated?
try another lot of temed.
give a longer time to polymerize ("age" the gel) to ensure complete polymerization.
prerun the gel to remove contaminants that may affect banding (eg-acrylic acid from acrylamide decomposition, leftover aps).
since it had been working until recently, what did you change?
I think 6% (29:1) is sufficient for resolution of DNA fragments in that range.
even the water quality you used change the polymerization of your gel. I had a bad experience with the change of pH of dH20 which i always used. Are the samples enter the gel or some of them gathered on the top of the gel? And how long have you been waiting for the gel to polymerize?