Hello Everyone,
I have another doubt regarding my RNA sample which 260/280 is quite fine which shows 1.7 to 1.9 value and it shows good band omn agarose gel but bit layer of smear which might be due to impurity.
We are getting 260/230 value in between 1.0 to 1.5 which is principally does not satisfy the purity level of RNA.
We have dissolved our RNA in 20 micro litre DEPC water and again we want to purify our RNA to increase its purity level.
What we can do for this improvement?
Kindly give your suggestion
Hi Gupta,
If you are using organic extraction method, I suggest you should take more precaution during transferring between phase (upper layer, interphase and lower layer). Because from my experiences, this step easily affect the ratio.
Another procedure that can be your option is by concentrating your RNA using RNA concentrator kit. Maybe you can decrease your RNA volume from 20uL to 15 or 10uL. Definitely, you can dilute back to get your desired concentration. This method may improve your ratio value.
Thanks, hopefully this help.
Hi,
What is the protocol? If you use Trizol method, in the alcohol step, I suggest you to wash the RNA with alcohol 75% twice, I tested this and the 260/230 valeu increased to 1.9.
Think about for second. Did you do any poly-a tailing since it is normal to see some smearing after tailing step.
Increase the volume to 400 microliters and an additional ethanol precipetation, preferentially with ammoniumacetate will do the trick. Dissolve the RNA in a buffered solution to prevent autohydrolysis
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA