Biology Reference
In-Depth Information
13. 1× TEN buffer: 10 mM Tris-HCl pH 8.0, 1 mM EDTA,
100 mM NaCl.
14. Tube rotator.
1. RNaseOUT recombinant ribonuclease inhibitor.
2. Dry bath and electrophoresis system: Same as above.
3. Gelbond PAG film (Lonza).
4. Gel dryer, cold trap, and vacuum pump.
5. Storage phosphor screen.
6. Phosphorimager such as the Typhoon 9410 Variable Mode
Imager (GE Healthcare).
7. Image analysis programs such as Quantity One (Bio-Rad),
ImageQuant (GE Healthcare), or ImageJ (NIH, free online).
2.4 Reconstituted
pri-miRNA Processing
Assays
3
Methods
1. Transform the NC1 expression plasmid to
E. coli
. Spread the
bacteria on an LB agar plate with appropriate antibiotic and
incubate at 37 °C overnight.
2. Inoculate a culture containing 150 mL of LB medium and
appropriate antibiotic with a single colony. Shake at 250 rpm
and 37 °C overnight.
3. Inoculate the desired volume of LB medium with antibiotic for
overexpression. Shake at 250 rpm and 37 °C until OD
600 nm
reaches 1.0-1.2 as measured using a spectrophotometer
equipped with a turbidity cuvette holder (
see
Note 3
).
4. Induce NC1 expression by adding IPTG and δ-ALA, both to
a final concentration of 1 mM (
see
Note 4
). Continue to shake
for 3.5-4 h at the same temperature. Collect the cells by cen-
trifuging at 5,000 ×
g
at 4 °C for 15 min. The pellets should
have a noticeable brown color.
5. Store the pellets at −80 °C.
3.1 Expression of
Heme-Bound DGCR8
NC1 in E. coli
3.2 Purification
of NC1
1. Completely resuspend cell pellet in ice-cold DGCR8 lysis buf-
fer (40 mL per L of culture).
2. Sonicate the cell suspension using the ultrasonic processor at
80 % power, 1-s on and 1-s off, for a total of 7-8 min. To avoid
overheating the lysate, a 30-s break is taken after each minute
of sonication and the container is kept on ice at all times.
3. Centrifuge the lysate at 45,000 ×
g
for 30 min at 4 °C.
4. Load the supernatant onto a HiTrap SP column equilibrated
with DGCR8 buffer
A
. Elute using a linear gradient of DGCR8
buffers
A
and
B
. The protein elutes at around 300 mM NaCl
