I have isolated total RNA from u2os (human) cell line. All times my 28 RNA band was not sharp from 18s RNA it got degraded. Anyone have suggestions?
If you are using kit please follow striclty the manufacturer's recommendations. And try to be as quick and effecient in executing the protocol as you can. Make sure that all your micropipettes are deignated for work with RNA isolation only and all your filter tip, microfuge tubes, plastic and glasswares are RNase-free. Treat your bench area with RNasedeactivating solutions first such as EDTA/NaOH follwoed by DEPC-treated water prepared at a final conc of 0.1% and had been autoclaved. Always minimize the time between sample collection and addition of lysis buffer. Better to work with fresh samples and if you have to stores your tissues you must snap-freeze them in liquid N2 as quickly as you can and store them at -80°C untill further use. You might alternatively dip your tissues in RNA-inhibiting solutions such as RNALater which is quite effective and you can store it as such in the normal fridge for a few days before you actually extract them. If you are working with a traditional way make sure that all your reagents autoclaved, filter-sterilized, and prepared using DEPC-treated water and all other roles applies and always work on ice until you finally elute or suspend your isolated RNA in buffer or water. Best o luck. mourad
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