Isolation of total RNA using CsCl/EtBr gradients--密度梯度法提取总RNA

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

80μl 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 μl 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