@RNA for S1 or PE analysis must be free of chromosomal DNA. This can be accomplished by extraction of the RNA with hot phenol, but phenol can, especially if its pH is acidic, cause the specific loss of certain RNAS, i.e.polyadenylated RNAs. This method, which is simply lysis by Frech pressing in RNase inhibitor-containing buffer followed by phenol:chloroform extraction, EtOH precipitation & CsCl/EtBr isopycnic centrifugation, is relatively rapid, easy, & results in an undegraded RNA preparation essentially free of DNA (without the use of DNase). Because the procedure does not rely on cell wall hydrolytic enzymes (lysozyme) or any other specific properties cell envelop, RNA from almost any type of cell can be isolated.
@Remember to use RNase-free technique throughout this procedure. Use DEPC-treated ddH2O, & absolute EtOH. And remeber - Ethidium bromide is MUTAGENIC! Wear gloves & clean up after yourself.
@A Ti50 or Ti75 rotor can also be used - more tubes will, of course, be required.
- Reagents
- STE-SDS
| 100mM |
NaCl |
| 50mM |
Tris, pH 8 |
| 1mM |
EDTA |
| 0.1% |
SDS |
- CsCl/EtBr sol'n
dissolve for each ml of STE/CsCl, add 80ul 10mg/ml Ethidium Br
- Saturated 2-PrOH
Mix equal volumes of:
- isopropyl alcohol
- 5M NaCl, 50mM Tris, pH8, 1mM EDTA
Shake vigorously, & allow to settle. Use the upper, organic phase.
- SSI Lysis buffer (per 200ml)
|
|
final conc. |
working conc. |
| 1.9380 grams |
Tris,pH 7.5 |
80 mM |
35.6 mM |
| 0.4067 grams |
MgCl2-7H2O |
10 mM |
4.4 mM |
| 140 ul |
2-mercaptoethanol |
10 mM |
4.4 mM |
SSI mix is prepared by mixing equal volumes of SSI lysis buffer & SSI lytic mix.
- Methods
- Grow 2liters, to mid-log phase, of strain from which RNA will be isolated. Harvest by centrifugation at 5KRPM, 4C 10 min. (GSA or GS3 rotor).
- Resuspend the cells in 20ml SSI mix & French press at 20000 PSI.
- Mix the lysate with 20ml Phenol:chloroform. Vortex & centrifuge 10KRPM, 4C 10 min. (HB4 or SS34 rotor), then collet the upper, aqueous, phase by pipette.
- Repeat step 3 twice more.
- Add 1/10th volume of 3M Na acetate and 2 volumes of EtOH. Incubate for 1hr at -70, then centrifuge at 10KRPM at 4C for 20 min. (SS34 or HB4 rotor). Discard the liquid & drain the pellets dry of EtOH.
- Dissolve the RNA in 20ml STE-SDS, then carefully remeasure the volume of the mixture. 0.11 volumes of 2mg/ml ethidium bromide, then add 1g of CsCl for each ml of the RNA/EtBr solution, and thoroughly dissolve.
- Load the solution into a sealable Ti60 tube. Top off the tube with blank CsCl/EtBr sol'n, & seal. Load the tube (and a balance) into the Ti60 rotor and centrifuge for 2 days at 38K, 20C.
- Collect the DNA band via syringe through the tube (don't forget to pucture the top of the tube first) if desired. Cut the tube in half, and drain the CsCl/EtBr sol'n off of the RNA pellet.
- Dissolve the RNA (it may take some work) in ddH2O, then add 1g of CsCl for each ml of RNA sol'n.
- Add an approximately equal volume of Sat'd 2-PrOH, mix, & allow to resettle. Collect the upper phase & discard. Repeat until no pink color remains in either phase, then repeat twice more to be sure all of the ethidium bromide is gone.
- For each ml of CsCl/RNA sol'n, add 2ml of H2O and 9ml of EtOH. Incubate overnight at -20C.
- Centrifuge 20min, 10KRPM 4C (HB4 or SS34 rotor), & discard the supernatant. Drain the RNA pellet dry & dissolve in 1ml ddH2O.
- Remove a small aliquot (20ul) for analysis in a minigel (2% agarose). Store the RNA at -20C.