A variety of RNA isolation procedures are available for different species of eubacteria - it is useful, however, to have a "general" method for isolating RNA from any specie you start to work with, such as the procedure described here. This procedure generally yeilds 20-50mg of RNA at good purity. No effort is made in this procedure to eliminate DNA, which is a minor contaminant. This procedure is primarily useful to those working with stable RNAs, or others needing large quautities of RNA. Rapidly turned-over RNAs should be isolated from RNA isolated by a more rapid procedure, such as the single-step RNA isolation procedure of Gopalakrishna, et al.
- Materials
- STE-SDS (for STE, omit SDS)
| 100mM |
NaCl |
| 10mM |
Tris, pH8 |
| 1mM |
EDTA |
| 0.1% |
SDS |
- Lytic solution
| 10% |
sucrose |
| 10mM |
Tris, pH8 |
| 5mM |
EDTA |
- Triton solution
| 2% |
Triton X-100 |
| 50mM |
Tris, pH8 |
- Lysozyme solution
10mg/ml lysozyme in Lytic solution
- 250mM EDTA
- 3M NaOAc (sodium acetate)
- Sat'd phenol
add an equal volume of 1M Tris (pH8) to some phenol, shake well, then allow to settle. Discard the upper phase (aqueous) & add an equal volume of TE. Mix well, & store at 4C.
- Methods
- Grow up enough of the organism to late log to get about 20g wet weight of cell paste (try 4 liters). (The procedure can be used with less paste by scaling down.) Cool the cells on ice, then pellet by centrifugation at 5KRPM , 10min., 4C (GS3 or GSA rotor), wash in 100ml STE or saline and recentrifuge. Discard the supernatants.
- Resuspend the cells in 20ml lytic solution, add 4ml lysozyme sol'n, mix, & incubate 5 min. on ice.
- Add 8ml 250mM EDTA, mix, & incubate on ice 5 min.
- Add 30ml triton solution, mix, & incubate 20 min. on ice.
- French press the suspension at 20KPSI, and remove cell debris by centrifuging at 10KRPM, 4C,10min HB4 rotor. Collect the supernatant.
- Add an equal volume of sat'd phenol, mix vigorously, and centrifuge 10KRPM, 4C, 10min (HB4). Collect the upper (aqueous) phase. Repeat twice more.
- Add 0.1 volume of 3M NaOAc and 2.5 volumes of EtOH & incubate overnight at -20C. Collect the RNA by centrifugation at 7KRPM, 20 min., 4C (GSA or GS3 rotor), drain the pellet dry, and dissolve in 25ml STE-SDS. Precipitate by adding 3ml 3M NaOAc and 75ml EtOH, incubate at -70 for 1hr, the repellet 10KRPM, 4C, 20min., (SS34 or HB4 rotor), drain and dry the pelletes.
- Dissolve the RNA in 10ml STE-SDS. Measure the absorbance of a 1:100 dilution of the sample & calculate the RNA concentration assuming that an A260 of 1 = 40ug/ml RNA.