Hi everyone! I would like some help about purified RNAextraction using the PureLink RNA MiniKit. I followed the protocol that the company suggests for 800000 cells. I had 399ng/μl concentration and 260/280=2.05 but 260/230=1.07. Also, OD260=9.9 and OD280=4.87(nanodrop). I would like some help for these odd numbers of OD 260 or 280. Should I always check these OD or I care only for the ratio?
Thank you in advance for your help!!!!
You may need to contact the manufacturer for assistance.
You have a good concentration of RNA, the A260/280 ratio looks good (little inverted, but not bad). The A260/230 isn't terrible. You may have some solvent or ethanol coming through the membrane. Check your desalting and wash buffers.
That said, your absorbances are very high (you'll start losing fidelity around 2). Dilute your sample 1-->10 (or 1-->20) (TE buffer, pure water, whatever) and multiply the final concentration by dilution.
Don't be freaked out if your corrected value isn't showing 399 ng/uL. As I said, absorbance around 2 or higher starts getting harder for the machine to detect differences.
Good luck!
- Agreed. The absolute values do not matter
- Your ratios are excellent. That is all that matters !
You always care the ratio.
Every single OD is meaningless alone.
Good job.
Good luck
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