Biology Reference
In-Depth Information
4. Load the supernatant onto a Ni Sepharose High Performance
column. Wash the column extensively using the Drosha wash
buffer, and elute the His
6
-Drosha
390-1374
protein in the Drosha
elution buffer.
5. Dialyze the purified His
6
-Drosha
390-1374
against the Drosha
storage/reaction buffer.
6. Aliquot in 10 μL per tube, freeze in liquid nitrogen or dry
ice-ethanol mix, and store in −80 °C freezer.
1. Set up the transcription reaction by adding the following:
10 μL H
2
O.
2 μL 10× transcription buffer.
2 μL 10× NTP mix.
2 μL DNA template.
2 μL [α-
32
P] UTP.
2 μL T7 RNA polymerase (always add last).
Incubate the transcription reaction at 37 °C for 2-3 h.
2. Add 20 μL 2× RNA loading dye to the reaction.
3. Pour a denaturing 15 % polyacrylamide gel (1× TBE, 7 M
urea). Assemble the gel sandwich. Induce polymerization of
the 50 mL gel mix by adding 50 μL TEMED and 500 μL 10 %
APS. Pour the gel, insert the comb, and let stand at room
temperature for 1 h. Mount the gel on the electrophoresis
apparatus. Pre-run the gel in 1× TBE at a constant power of
12 W for 20 min.
4. Load the transcription onto the gel. Run the gel at 12 W until
the bromophenol blue reaches the bottom of the gel.
Disassemble gel sandwich, and leave the gel attached to one
glass plate.
5. Cover the gel with a plastic wrap. Expose the gel to an autora-
diography film. Excise out the band. Expose the gel to another
film to confirm the excision.
6. Crush and soak the gel piece in 1× TEN buffer at 4 °C for at
least 10 h.
7. Precipitate the RNA by adding 3 volumes of ethanol and 0.1
volume of 3 M sodium acetate pH 5.2. Resuspend the RNA in
H
2
O.
8. Determine radioactivity using scintillation counting. Dilute
the RNA to ~10,000 cpm/μL in water prior to the processing
assay.
3.4 Transcription
and Purification of
pri-miRNAs Uniformly
Labeled with
32
P
3.5 pri-miRNA
Processing Assay
1. Set up a 10-μL processing reaction by mixing the following (in
the order shown):
