I have been working on Red blood cell-derived extracellular vesicles as Antisense Oligonucleotide (ASO) carriers. We normally run agarose gel to quantify the loading efficiency. I used naked ASO at different concentrations as standard. Recently we encountered a problem of ASO degradation on agarose gel quickly. If I stop the running after 5 min i still could see the bands from my ASO standard dilutions, or even my loaded EVs, but if I run longer, they start to disappear one by one from lowest to the highest concentration standard bands. I run the gel at 100 volts. We suspected the contamination of RNAse as our ASOs are gapmers, so we cleaned everything with RNa zap but it still happened. Could anyone help with some opinions and suggestions? Thank you!!
Could it be that short single stranded dna will diffuse readily in agarose gels and the disappearance is simply diffusion to the point where the band becomes invisible as it runs further through the gel. If so run a higher concentration gel slowly in a cold room or fridge to minimise thermal diffusion or possibly run PAGE with silver staining
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