Hi everyone, recently I am too much worry about my RNA which is always bad in sense of genomic DNA or very big problem is degradation of RNA. I try many times, I always get high concentrated RNA when check on nanodrop of about 2000 to 3000 ng/ul (260/280= 1.9 and 2) but when ever i run gel my RNA seems terrible. I always use RNase free tips, pipettes, glassware etc. Please give me some suggestions. I want to ask when i wash pellet with ethanol is it important to centrifuge it? Is centrifugation cause RNA degradation? Please tell me some critical steps during RNA extraction. Thanks
centrifugation With ethanol is as usual procedure for RNA extraction,Storage and handling of sample is an important step, are you stored your samples in RNA later (in -80) .Few more thing after chloroform vigorous shaking is an important step and pink color will be observed for good RNA extraction, another thing after isoprop a white buffy ring is an essential between two solution interface, don't over dry the RNaA pellet, and dissolve in RNA storage solution.
I store RNA at -80 after check its quality & quantity. Yes pink & white phase see very clearly. Normally i dry pellet for 15 min and store in DEPC water.
Picture please.
Do you run a denaturating gel?
Did you check for phase invasion?
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