after adding Isopropanol, and centrifuging, I’m not seeing visible white pellet at the bottom of the EP tube, is that means I proceed wrong ? please need some help !
Dear Khadidja Ouenzar, Here are a few possibilities to consider:
A. Measurement limitations, In some cases, pelleting bacterial RNA using the Trizol method may not result in a visible pellet due to its small size or low density. In such cases, you can confirm the presence of RNA by using alternative techniques such as measuring RNA length using gel electrophoresis.
B. Sample quality, If the sample contains various contaminants or different compounds, it may interfere with the formation of a visible white pellet. Ensure accuracy in sample collection and preparation, and make sure your sample is clean and pure.
C. Centrifugation method, Be careful to use the correct settings for centrifugation. If the speed and duration of centrifugation are set incorrectly, it may not have the desired effect on separation.
D. Operational errors, Make sure you have followed each step of the protocol correctly. Mistakes in chemical preparation and mixing and errors in timing or temperature can impact the results.
Try soaking the pellet (or region of where the pellet would be) in ice cold 80% ethanol. RNA pellets that come from precipitations using isopropanol tend to be clear. Soaking will allow the pellet to become white. Just note, when precipitating with isopropanol, your pellet will contain more salt. You will need to wash more.
Without details of the protocol you performed, it is difficult to troubleshoot what exactly happened. But try my suggestion above to see if any RNA pellet appears.
Thank you for your replies, i will resume the experiment and considerate your suggestions 🙏
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