I extract the RNA from muscle tissue using Qiagen RNeasy Micro kit (50) (kat.74004). Everything fine with RNA concentration - about 300-500 ng/microliter. Before putting it into wells I mix my RNA with equal amount of 2x loading dye (thermoscientific R0641). The concentration of agarose gel - 1.2%, but I have tried to use 1% and 2% - the result is always the same. Voltage is 5 Volt per centimeter (which equals 65V for entire gel). My wells are fine (you also can see it on the picture). I add 0.5 µg/ml ethidium bromide both to gel and TBE buffer. I prepare my TBE buffer using next components: Tris, boric acid, Liquid EDTA + H2O. I use protocol in whic the next steps are needed: Heat samples and ladder for 10 min at 70°C. 4. Chill samples and ladder on ice for 3 min and spin briefly prior to loading. So What CAN BE WRONG and why I get two, let`s say, drops of RNA on two sides of wells rather than line? There are RiboRuler Ladder in the first well.
using pipette wash off the salts in the wells. just up-down several times.
Don't heat your RNA samples! What you are seeing is badly degraded RNA. You need to keep your samples on ice at all times.
Katie A Burnette Hello. Do you mean that I should even skip 15 minutes heating step? Or just keep my samples on PCR-cooler (like on picture) all the time before and after that steр?
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