Recently BsB author Yevgeniy Grigoryev shared a total RNA isolation protocol. The one I use is even simpler – no expensive Trizol, which is a mix of phenol and some salts, all that is required is some Tris, SDS and phenol/chloroform mix. I have never used this protocol on non-yeast cells but I am almost sure that it can be applied to any cell type after the homogenisation step in the RNA buffer. Changing the buffer pH from neutral to acidic – pH 4.5 – will allow you to isolate aminoacylated tRNAs as well.
1. Grow 25 – 100 ml of cells to OD 600 0.25 – 0.5 (You don’t even need a spectrophotometer for this).
2. Spin cells, wash them in 1 ml dH2O and transfer to a screw-cap tube. You can snap-freeze pellet at this stage.
3. To 1 volume of cold RNA buffer add SDS to final concentration 0.5% (1/40 volume 20% SDS).
4. Resuspend frozen pellet in 200µl cold SDS/RNA buffer.
5. Add 1 volume phenol/chloroform; fill with beads to reach above solution.
6. Break open (usually 30 sec/ 1 min on ice/30 sec on maximum Ribolyser setting) but your routine breakage procedure can be just as effective.
7. Fill tube with RNA buffer (no SDS added), vortex, and spin 5 min keeping cold.
8. You will see phenol/chloroform fraction at the bottom of the tube, white layer of debris and top buffer level. Take the top level and transfer to a fresh RNAse free eppendorf.
9. Extract aqueous fraction with phenol/chloroform twice, shaking for 5 min. Add 0.9 – 1 volume of isopropanol, mix, and spin at RT 15 – 20 min. Because the buffer contains a lot of salt, no additional sodium acetate is necessary
(If 1/10 3M sodium acetate is accidentally added, get rid of the salts by dissolving the dried pellet in 600 µl RNA buffer, incubating 5 min with shaking, adding 600 µl of isopropanol and spinning 10 – 15 min. After this go to step 9).
10. Wash pellet with 70%, air dry, resuspend in 30 – 50 µl dH2O or TE and measure OD260/280 ratio.
RNA buffer (50 ml)
· 100 mM EDTA pH8.0 (10 ml 0.5M stock)
· 100mM NaCl (1 ml 5M stock)
· 50 mM Tris-HCl pH8.0 (2.5 ml 1M stock)
· 36.5 ml dH2O
For Easy Yeast DNA – both genomic and plasmid – isolation see this article.
Have you any tips for inexpensive nucleic acid isolation in the lab?