DEP treatment solutions. Add 0.3 ml lysing buffer (0.1 M Hepes, 0.2 M EDTA, 10% SDS, 0.02 M EGTA (pH 7.5) [DEP-treated) to 3 ml bacterial solution. Extract with 1 vol 1000:140:0.4 (w:w:w) phenol:m-cresol:hydroxyquinoline. Add directly to 2 volumes ethanol (~6.6 ml). Precipitate for 16 hrs at -20 ℃.
Buffers needed:
A. 0.2 M MOPS (pH 7.0), 50 mM sodium acetate, 1 mM EDTA.
B. Formaldehyde, 37%
C. Formamide - deionized till pH 7.0 and recrystallized at 0 C, stored at -20 ℃.
D. 50% glycerol, 1 mM EDTA, 0.4% bromphenol blue, 0.4% xylene cyanol.
PREPARATION OF VANADYL-RIBONUCLEOSIDE COMPLEXES WHICH INHIBIT RIBONUCLEASE ACTIVITY. reagents: vanadyl sulfate adenosine cytidine guanosine uridine 10 N NaOH
method:
For 100 ml vanadyl complex,
Make 10 ml 2 M vanadyl sulfate (0.366 g/ml) To make 80 ml H2O in a 250 ml flask, add 1.34 g adenosine, 1.45 g guanosine, 1.25 g uridine, and 1.25 g cytidine = 5 mM XRs Boil in H2O water bath with N2 sparge (XRs dissolve but ppt. upon cooling). Add vanadyl sulfate and continue to boil and sparge 1 min (solution should remain blue). Turn off H2O bath, continue to sparge, and add 8 ml 10 N NaOH, then 4 ml 1 N NaOH (ugly grey ppt.; redissolve when all NaOH added) check pH = 7-9 range. Resulting 0.2 M vanadyl nucleoside solution stored at -20 ℃. Upon thawing, redissolve ppt. at 65℃ (not above).
RNA preparation and electrophoresis (p202-203)
