i have RNA that i extracted it with trizol. it has good concentration ( 1010ng/ul) and good 260/280 (1.9) but 260/230 isn't good ( 0.5)....now how can i improve it? can i use again from this RNA to improve 260/230 or i must extract again?
Laraib Urooj
260/230 ratio being the measure of purity should be higher than 260/280. ower values as in your case is the result of contamination as contaminants(polysaccharides) absorb at 230. you need to re precipitate with sodium acetate or ethanol.
Its better to redo the experiment because washing will give you small amount of RNA.
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Laurence Stuart Dawkins-Hall
- Generally speaking 260/230 ratios < 1.0 and especially ~ 0.5 are caused by residual salt in your sample which in turn might come from GITC in the lysis buffer
- Thus, the answer is to desalt your sample
- To do this you can re ppt your sample with 1/20 3M Sodium acetate pH 4-5 plus 3 volumes of ethanol and incubate at -20C for 1 hour (or over night if more convenient)
- Then spin down and wash the resulting pellet x 2 with ROOM temp 70% ethanol (by vortexing for 1 minute; spinning for 5 min to pellet again; removing 70% ethanol; vortexing for another minute; spinning for 5 minute and then take off this second 70% ethanol aliquot before air drying for 15 minutes and resuspending)
- Vortexing and use of ROOM temp 70% ethanol leads to more effective removal of salt which in my experience will bring your 260/230 ratio >/= 1.0
- After Trizol and chloroform extraction by the way did you re precipitate with an equal volume of isopropanol at room temp; spin and then wash the pellet with 70% ethanol ?
- If you did I would suggest in future you follow your Trizol chloroform extraction and isopropanol ppt with 2 x room temp 70% ethanol washes to maximise desalting and thus keep your 260/230 ratio > 1.0
- Your Trizol and Chloroform extractions are designed to keep your 260/280 ratio ~ 1.9-2.0 (by removing protein and lipid) where as the wash step is designed to desalt which is indicated by your 260/230 ratio
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