- 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 C.
RNA preparation and electrophoresis (p202-203)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 C.
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 C.
- Upon thawing, redissolve ppt. at 65 C (not above).
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提取棉花RNA--RNA EXTRACT