3.6 Internally
Labeled Klenow Probe
(Fig. 4a)
In order to generate more sensitive probes, we recommend using
the Klenow labeling procedure. Such probes detect multiple siRNAs that arise in clusters—e.g., from the long terminal repeats of a
retrotransposon (Fig. 3). Prior to probe radiolabeling, design PCR
primers that flank a genomic region of interest. Amplify this region
using Taq polymerase and purify the PCR fragment from an agarose gel. 100 ng of pure PCR fragment will be used for each
Klenow probe preparation.
1. In a 1.5 mL safe-lock tube, prepare:
PCR fragment
100 ng
Random hexamer primer (100 μM)
1 μL
Milli-Q water
Fill to 9 μL
2. Heat the mixture to 95
C for 5 min.
3. Transfer the tube immediately to ice for at least 5 min.
4. Add the following components to the same tube (see Note 3):
10Â Klenow buffer
2 μL
dATP/dGTP/dTTP mix (1 mM each)
3 μL
Klenow fragment enzyme
1 μL
[α
32 P]-dCTP (~50 μCi)
5 μL
5. The labeling reaction is performed for 45 min at 37
C.
6. The radiolabeled probe is then purified on an Illustra MicroSpin G-25 column:
Prepare the column by vortexing, followed by centrifugation at 800 Â g for 1 min. Transfer the column to a clean
1.5 mL tube and load the sample. Centrifuge for 2 min at
800 Â g. Radiolabeled DNA fragments are in the flowthrough.
7. Denature this probe by heating to 95
C for 3 min and then
transfer it to ice for 5 min.
8. Pipette the probe into the tube with the pre-hybridized membrane (from Subheading 3.5). Do not touch the membrane
with the pipette, or let the concentrated probe land directly on
the membrane surface.
9. Hybridize overnight. Conditions are specified in Subheading
2.4.
Genome-Scale and Northern Blot Analyses of siRNAs
403
Labeled Klenow Probe
(Fig. 4a)
In order to generate more sensitive probes, we recommend using
the Klenow labeling procedure. Such probes detect multiple siRNAs that arise in clusters—e.g., from the long terminal repeats of a
retrotransposon (Fig. 3). Prior to probe radiolabeling, design PCR
primers that flank a genomic region of interest. Amplify this region
using Taq polymerase and purify the PCR fragment from an agarose gel. 100 ng of pure PCR fragment will be used for each
Klenow probe preparation.
1. In a 1.5 mL safe-lock tube, prepare:
PCR fragment
100 ng
Random hexamer primer (100 μM)
1 μL
Milli-Q water
Fill to 9 μL
2. Heat the mixture to 95
C for 5 min.
3. Transfer the tube immediately to ice for at least 5 min.
4. Add the following components to the same tube (see Note 3):
10Â Klenow buffer
2 μL
dATP/dGTP/dTTP mix (1 mM each)
3 μL
Klenow fragment enzyme
1 μL
[α
32 P]-dCTP (~50 μCi)
5 μL
5. The labeling reaction is performed for 45 min at 37
C.
6. The radiolabeled probe is then purified on an Illustra MicroSpin G-25 column:
Prepare the column by vortexing, followed by centrifugation at 800 Â g for 1 min. Transfer the column to a clean
1.5 mL tube and load the sample. Centrifuge for 2 min at
800 Â g. Radiolabeled DNA fragments are in the flowthrough.
7. Denature this probe by heating to 95
C for 3 min and then
transfer it to ice for 5 min.
8. Pipette the probe into the tube with the pre-hybridized membrane (from Subheading 3.5). Do not touch the membrane
with the pipette, or let the concentrated probe land directly on
the membrane surface.
9. Hybridize overnight. Conditions are specified in Subheading
2.4.
Genome-Scale and Northern Blot Analyses of siRNAs
403