Biology Reference
In-Depth Information
Fig. 2
15 % Denaturing PAGE of (
a
) circular ssDNA template and (
b
) linear
5
′
-phosphorylated ssDNA (SYBR Gold stain,
λ
ex
= 470 nm,
λ
em
= 535 nm)
2. Dilute Dicer (1 U/
L.
3. Use an aliquot of diluted Dicer to prepare heat-denatured
Dicer for control reactions. Heat to 90 °C for 15 min, and
store at −20 °C.
4. In a clear 96-well plate, 20-
μ
L) with Dicer dilution buffer to 0.04 U/
μ
L reactions were performed in at
least three replicates per test compound (
see
Notes 10
and
11
). Calculate the volume needed for all reactions, and prepare
a pre-miRNA dilution (71.4 nM) in Dicer buffer. For ten reac-
tions, for example, dilute pre-miRNA (11
μ
μ
L, 1
μ
M) in Dicer
L, 1×).
5. Add the test compound in the desired concentration or solvent
(4
buffer (143
μ
L) into each well. Mix by repeated
aspiration, and incubate at RT for 30 min.
6. Add Dicer (2
μ
L) and pre-miRNA (14
μ
L) to each well and mix by repeated aspiration.
Incubate at 37 °C for 2 h in a thermocycler with heated lid.
Increase temperature to 90 °C for 15 min for inactivation, and
store on ice.
μ
3.4 Rolling Circle
Amplifi cation
1. Towards the end of the Dicer incubation, prepare a master-mix
solution for the BRCA reaction on ice. Again consider all reac-
tions including all controls. The volume that is added to each
BRCA reaction should be 10
μ
L, including 2× ThermoPol
buffer, 0.4 mM dNTPs, 0.4
M secondary primer, 1:10,000
(v/v) SYBR Gold, 10 % DMSO, and 4 nM circular ssDNA
template.
μ
