Hi all
Can someone tell me what I'm doing wrong with my gel shifts? My RNA
probes sometimes stay in the wells rather than migrating, my results (an
X-ray of the gel) show only tiny complexes of variable sizes when I know
I should get large complexes with this particular RNA (3'UTR of mouse
protamine 1). Also, my X-rays show these same tiny complexes in lanes
containing only RNA probe and RNase T1. Help!
Allison Leahy
PS: for anyone who wants to see my protocol:
Protein extract prep:
dissect testes from 2 mice. wash testes 3 times with buffer A (10 mM
HEPES (pH 7.6), 1.5 mM MgCl2 , 10 mM KCl, 0.5 mM DTT, and 1/4 tablet of
protease inhibitor cocktail tablets ( Boehringer Mannheim) ). Resuspend
testes in buffer A to a final volume of 1.0 ml/g of tissue. Homogenize
with 20 strokes of a Dounce homogenizer and B pestle. Centrifuge tissue
at 14,000 rpm for 2 minutes. Mix supernatant with 0.11 volumes buffer B
(0.3 M HEPES (pH 7.6), 1.4 M KCl, 0.03 M MgCl2). Centrifuge at 31,000
rpm for 20 minutes in an ultracentrifuge. Draw off supernatant in 20 ul
aliquots and flash freeze it. Do a Bradford assay to determine
concentration. Seek 25-50 mg/ml
RNA probe prep:
Digest DNA in 100 ul volume. Extract 2 times with phenol. Precipitate
with ethanol. Wash with 0.5 ml 75% EtOH (DEPC treated). Resuspend
pellet in 20 ul TE80 buffer. Make RNA probes according to instructions
on Promega kit: 4 ul of 5x buffer, 2 ul of 100 mM DTT, 0.5 ul RNasin, 1
ul nuclease free water, 1 ul 10 mM rATP, 1 ul of 10 mM rCTP, 1 ul of 10
mM rGTP, 2.4 ul of 100 uM rUTP, 0.2 ug template DNA, 5 ul (50 uCi)
32P-UTP, and 1 ul polymerase (I use T7, SP6, and T3, depending on the
particular DNA I'm using). Incubate at 37oC for 1 hour. Add 1 ul RQ1
DNase and incubate for 15 minutes at 37oC. Add DEPC water to 80 ul. Add
80 ul phenol, vortex, and centrifuge 2 minutes. Add DEPC water to a
volume of 100 ul. Run probe through a Sephadex G-50 column that has
been prewashed with 2 ml DEPC water. Ethanol precipitate using DEPC
treated 5M NaCl. Wash with 75% ethanol (DEPC treated), and resuspend in
50 ul DEPC water. Run 5 ul through a scintillation counter to determine
cpm. Dilute to a concentration of 100,000 cpm/ul
Binding reaction:
Put 1 ul probe in Eppendorf tubes. Incubate at 70oC for 5 minutes to
denature. Add 5 ul RNA binding buffer (preheated to 70oC) (40 mM HEPES
(pH 7.6), 6 mM MgCl2, 80 mM KCl, 4 mM DTT, 10% glycerol), and 3 ul DEPC
treated water (also preheated). Incubate 30 minutes at room
temperature. Add 1 ul protein extract to appropriate tubes and incubate
at room temperature 20 minutes. Add 1 ul RNase T1 to appropriate tubes
and incubate at room temperature 10 minutes. Add 2 ul heparin to
appropriate tubes and incubate at room temp 10 minutes. The end result
should be for every different probe, there are 5 tubes: 1)containing
probe; 2) containing probe and RNase T1; 3) probe and protein extract;
4) probe, T1, and extract; 5) probe, T1, extract, and heparin.
To all tubes add 5 ul of 50% glycerol with bromophenol blue. Run
samples through a 4% nondenaturing polyacrylamide gel at 150 volts for
approximately 90 minutes. X-ray the gel.
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