From jwbrown@crab   Wed Jan 15 02:45:08 1992
Subject: Isolation of total RNA using CsCl/EtBr gradients


     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.


STE-SDS
     100mM NaCl
      50mM Tris, pH 8
       1mM EDTA
     0.1%  SDS

CsCl/EtBr sol'n
   10ml STE-SDS
   10g CsCl - 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 lytic mix (per 200ml)
         2g  SDS                        1%          0.5%
         40ml 0.1M EDTA                20mM          10mM
         158mg 1,10-phenanthroline      4mM           2mM
         160mg heparin                400ug/ml      200ug/ml

SSI mix is prepared by mixing equal volumes of SSI lysis buffer & SSI 
lytic mix.



1. 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).

2. Resuspend the cells in 20ml SSI mix & French press at 20000 PSI.

3. 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.

4. Repeat step 3 twice more.

5. 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.

6. 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.

7. 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.

8. 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.

9. Dissolve the RNA (it may take some work) in ddH2O, then add 1g of 
CsCl for each ml of RNA sol'n.

10. 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.

11. For each ml of CsCl/RNA sol'n, add 2ml of H2O and 9ml of EtOH.  
Incubate overnight at -20C.

12. Centrifuge 20min, 10KRPM 4C (HB4 or SS34 rotor), & discard the 
supernatant.  Drain the RNA pellet dry & dissolve in 1ml ddH2O.

13.  Remove a small aliquot (20ul) for analysis in a minigel (2% 
agarose).  Store the RNA at -20C.




JWBrown, unpublished data
Maniatis "Molecular Cloning" manual